Methods for selecting polynucleotides encoding T cell epitopes

ABSTRACT

The present invention relates to methods for the identification of antigens recognized by cytotoxic T cells (CTLs) and specific for human tumors, cancers, and infected cells, and the use of such antigens in immunogenic compositions or vaccines to induce regression of tumors, cancers, or infections in mammals, including humans. The invention encompasses methods for induction and isolation of cytotoxic T cells specific for human tumors, cancers and infected cells, and for improved selection of genes that encode the target antigens recognized by these specific T cells. The invention also relates to differential display methods that improve resolution of, and that reduce the frequency of false positives of DNA fragments that are differentially expressed in tumorous, cancerous, or infected tissues versus normal tissues. The invention further relates to the engineering of recombinant viruses as expression vectors for tumor, cancer, or infected cell-specific antigens.

The work reflected in this application was supported, in part, by agrant from the National Institutes of Health, and the Government mayhave certain rights in the invention.

INTRODUCTION

The present invention relates to novel methods for the identification ofantigens recognized by cytotoxic T cells (CTLs) and specific for humantumors, cancers, and infected cells, and the use of such antigens inimmunogenic compositions or vaccines to induce regression of tumors,cancers, or infections in mammals, including humans. The inventionencompasses methods for induction and isolation of cytotoxic T cellsspecific for human tumors, cancers or infected cells, and for improvedselection of genes that encode the target antigens recognized by thesespecific T cells. The invention also relates to differential displaymethods that improve resolution of, and that reduce the frequency offalse positives of DNA fragments that are differentially expressed intumorous, cancerous, or infected tissues versus normal tissues. Theinvention further relates to the engineering of recombinant viruses asexpression vectors for tumor, cancer, or infected cell-specificantigens.

BACKGROUND OF THE INVENTION

Current therapies for cancer include surgery, chemotherapy andradiation. The development and use of immunotherapeutic approaches,e.g., tumor targeting using antibody conjugates, “cancer vaccines”, etc.is an attractive alternative, but has, to date, met-with limited successfor a number of reasons. The development of monoclonal antibodiesspecific for tumor antigens, for example, has proved difficult, in part,because antigens that are recognized by monoclonal antibodies and thatare expressed by tumors and cancer cells are often also expressed bynormal, non-cancerous cells. In addition, the expression of membraneantigens targeted by antibodies is frequently modulated to permit growthof tumor variants that do not express those antigens at the cellsurface. A cell-mediated immune response may be more effective foreradication of tumors both because of the different array of effectorfunctions that participate in such responses, and because Tcell-mediated responses target not only membrane antigens but anytumor-specific intracellular protein that can be processed and presentedin association with major histocompatibility molecules. It is, for thisreason, much more difficult for a tumor to evade T cell-surveillance bymodulating membrane expression.

Immunotherapeutic approaches based on cell-mediated immune responses arelikely to be more effective, but antigens that are expressed by tumorsand recognized in cell-mediated immune responses are difficult toidentify and to produce. Development of an effective treatment forcancer through vaccination and subsequent stimulation of cell-mediatedimmunity, has remained elusive; the identification of effective antigensto stimulate cell-mediated responses has been successful only in specialcases, such as melanoma. In melanoma, the cytotoxic T cells (CTLs) thatmediate a cellular immune response against melanoma infiltrate the tumoritself, and such CTLs can be harvested from the tumor and used to screenfor reactivity against other melanoma tumors. Isolation of tumorinfiltrating lymphocytes has, however, not been a successful strategy torecover cytotoxic T cells specific for most other tumors, in particularthe epithelial cell carcinomas that give rise to greater than 80% ofhuman cancer.

To address the problem of identifying effective antigens for use invaccination, most previous work has focused on screening expressionlibraries with tumor-specific CTLs to identify potential tumor antigens.There are significant limitations to the existing methods of identifyingeffective antigens, including the excessively laborious and inefficientscreening process and the considerable difficulty in isolatingtumor-specific CTLs for most types of tumors.

Cancer Vaccines

The possibility that altered features of a tumor cell are recognized bythe immune system as non-self and may induce protective immunity is thebasis for attempts to develop cancer vaccines. Whether or not this is aviable strategy depends on how the features of a transformed cell arealtered. Appreciation of the central role of mutation in tumortransformation gave rise to the hypothesis that tumor antigens arise asa result of random mutation in genetically unstable cells. Althoughrandom mutations might prove immunogenic, it would be predicted thatthese would induce specific immunity unique for each tumor. This wouldbe unfavorable for development of broadly effective tumor vaccines. Analternate hypothesis, however, is that a tumor antigen may arise as aresult of systematic and reproducible tissue specific gene deregulationthat is associated with the transformation process. This could give riseto qualitatively or quantitatively different expression of sharedantigens in certain types of tumors that might be suitable targets forimmunotherapy. Early results, demonstrating that the immunogenicity ofsome experimental tumors could be traced to random mutations (De Plaen,et al., Proc. Natl. Acad. Sci. USA 85:2274-2278 (1988); Srivastava, &Old. Immunol. Today 9:78 (1989)), clearly supported the firsthypothesis. There is, however, no a priori reason why random mutationand systematic gene deregulation could not both give rise to newimmunogenic expression in tumors. Indeed, more recent studies in bothexperimental tumors (Sahasrabudhe, et al., J. Immunology 151:6202-6310(1993); Torigoe, et al., J. Immunol. 147:3251 (1991)) and human melanoma(van Der Bruggen, et al., Science 254:1643-1647 (1991); Brichard, etal., J. Exp. Med. 178:489-495 (1993); Kawakami, et al., Proc. Natl.Acad. Sci. USA 91:3515-3519 (1994); Boel, et al., Immunity 2:167-175(1995); Van den Eynde, et al., J. Exp. Med. 182:689-698 (1995)) haveclearly demonstrated expression of shared tumor antigens encoded byderegulated normal genes. The identification of MAGE-1 and otherantigens common to different human melanoma holds great promise for thefuture development of multiple tumor vaccines.

In spite of the progress in melanoma, shared antigens recognized bycytotoxic T cells have not been described for other human tumors. Themajor challenge is technological. The most widespread and to date mostsuccessful approach to identify immunogenic molecules uniquely expressedin tumor cells is to screen a cDNA library with tumor-specific CTLs(cytotoxic T lymphocytes). Application of this strategy has led toidentification of several gene families expressed predominantly in humanmelanoma. Two major limitations of this approach, however, are that (1)screening requires labor intensive transfection of numerous small poolsof recombinant DNA into separate target populations in order to assay Tcell stimulation by a minor component of some pool; and (2) with thepossible exception of renal cell carcinoma, tumor-specific CTLs havebeen very difficult to isolate from either tumor infiltratinglymphocytes (TIL) or PBL of patients with other types of tumors,especially the epithelial cell carcinomas that comprise greater than 80%of human tumors. It appears that there may be tissue specific propertiesthat result in tumor-specific CTLs being sequestered in melanoma.

Direct immunization with tumor-specific gene products may be essentialto elicit an immune response against some shared tumor antigens. It hasbeen argued that, if a tumor expressed strong antigens, it should havebeen eradicated prior to clinical manifestation. Perhaps then, tumorsexpress only weak antigens. Immunologists have long been interested inthe issue of what makes an antigen weak or strong. There have been twomajor hypotheses. Weak antigens may be poorly processed and fail to bepresented effectively to T cells. Alternatively, the number of T cellsin the organism with appropriate specificity might be inadequate for avigorous response (a so-called “hole in the repertoire”). Elucidation ofthe complex cellular process whereby antigenic peptides associate withMHC molecules for transport to the cell surface and presentation to Tcells has been one of the triumphs of modern immunology. Theseexperiments have clearly established that failure of presentation due toprocessing defects or competition from other peptides could render aparticular peptide less immunogenic. In contrast, it has, for technicalreasons, been more difficult to establish that the frequency of clonalrepresentation in the T cell repertoire is an important mechanism of lowresponsiveness. Recent studies demonstrating that the relationshipbetween immunodominant and cryptic peptides of a protein antigen changein T cell receptor transgenic mice suggest, however, that the relativefrequency of peptide-specific T cells can, indeed, be a determiningfactor in whether a particular peptide is cryptic or dominant in a Tcell response. This has encouraging implications for development ofvaccines. With present day methods, it would be a complex and difficultundertaking to modify the way in which antigenic peptides of a tumor areprocessed and presented to T cells. The relative frequency of a specificT cell population can, however, be directly and effectively increased byprior vaccination. This could, therefore, be the key manipulationrequired to render an otherwise cryptic response immunoprotective.

Another major concern for the development of broadly effective humanvaccines is the extreme polymorphism of HLA class I molecules. Class IMHC: cellular peptide complexes are the target antigens for specificCD8+ CTLs. The cellular peptides, derived by degradation of endogenouslysynthesized proteins, are translocated into a pre-Golgi compartmentwhere they bind to class I MHC molecules for transport to the cellsurface. The CD8 molecule contributes to the avidity of the interactionbetween T cell and target by binding to the α3 domain of the class Iheavy chain. Since all endogenous proteins turn over, peptides derivedfrom any cytoplasmic or nuclear protein may bind to an MHC molecule andbe transported for presentation at the cell surface. This allows T cellsto survey a much larger representation of cellular proteins thanantibodies which are restricted to recognize conformational determinantsof only those proteins that are either secreted or integrated at thecell membrane.

The T cell receptor antigen binding site interacts with determinants ofboth the peptide and the surrounding MHC. T cell specificity must,therefore, be defined in terms of an MHC: peptide complex. Thespecificity of peptide binding to MHC molecules is very broad and ofrelatively low affinity in comparison to the antigen binding sites ofspecific antibodies. Class I-bound peptides are generally 8-10 residuesin length and accommodate amino acid side chains of restricted diversityat certain key positions that match pockets in the MHC peptide bindingsite. These key features of peptides that bind to a particular MHCmolecule constitute a peptide binding motif.

Hence, there exists a need for methods to facilitate the induction andisolation of T cells specific for human tumors, cancers and infectedcells and for methods to efficiently select the genes that encode themajor target antigens recognized by these T cells in the properMHC-context.

Vaccinia Vectors

Poxvirus vectors are used extensively as expression vehicles for proteinand antigen, e.g. vaccine antigen, expression in eukaryotic cells. Theirease of cloning and propagation in a variety of host cells has led, inparticular, to the widespread use of poxvirus vectors for expression offoreign protein and as delivery vehicles for vaccine antigens (Moss, B.,Science 252:1662-1667(1991)).

Customarily, the foreign DNA is introduced into the poxvirus genome byhomologous recombination. The target protein coding sequence is clonedbehind a vaccinia promoter flanked by sequences homologous to anon-essential region in the poxvirus and the plasmid intermediate isrecombined into the viral genome by homologous recombination. Thismethodology works efficiently for relatively small inserts tolerated byprokaryotic hosts. The method becomes less viable in cases requiringlarge inserts as the frequency of homologous recombination is low anddecreases with increasing insert size; in cases requiring constructionof labor intensive plasmid intermediates such as in expression libraryproduction; and, in cases where the propagation of DNA is not toleratedin bacteria. Hence, there is a need for improved methods of introducinglarge inserts at high frequency, that do not require such laborintensive genetic engineering.

Alternative methods using direct ligation vectors have been developed toefficiently construct chimeric genomes in situations not readilyamenable for homologous recombination (Merchlinsky, M. et al., Virology190:522-526 (1992); Scheiflinger, F. et al., Proc. Natl. Acad. Sci. USA.89:9977-9981 (1992)). These direct ligation protocols have obviated theneed for homologous recombination to generate poxvirus chimeric genomes.In such protocols, the DNA from the genome was digested, ligated toinsert DNA in vitro, and transfected into cells infected with a helpervirus (Merchlinsky, M. et al., Virology 190:522-526 (1992);Scheiflinger, F. et al., Proc. Natl. Acad. Sci. USA 89:9977-9981(1992)). In one protocol, the genome was digested at the unique NotIsite and a DNA insert containing elements for selection or detection ofthe chimeric genomes was ligated to the genomic arms (Scheiflinger, F.et al., Proc. Natl. Acad. Sci. USA. 89:9977-9981 (1992)). This directligation method was described for the insertion of foreign DNA into thevaccinia virus genome (Pfleiderer et al., J. General Virology76:2957-2962 (1995)). Alternatively, the vaccinia WR genome was modifiedby removing the NotI site in the HindIII F fragment and reintroducing aNotI site proximal to the thymidine kinase gene such that insertion of asequence at this locus disrupts the thymidine kinase gene, allowingisolation of chimeric genomes via use of drug selection (Merchlinsky, M.et al., Virology 190:522-526 (1992)).

The direct ligation vector, vNotI/tk allowed one to efficiently cloneand propagate DNA inserts at least 26 kilobase pairs in length(Merchlinsky, M. et al., Virology 190:522-526 (1992)). Although, largeDNA fragments were efficiently cloned into the genome, proteins encodedby the DNA insert will only be expressed at the low level correspondingto the thymidine kinase gene, a relatively weakly expressed early classgene in vaccinia. In addition, the DNA will be inserted in bothorientations at the NotI site. Hence, there is a need for more efficientmethods of cloning large DNA fragments into the viral genome withaccompanying high levels of expression of the protein product encoded bythe DNA insert. There also-exists a need for improved direct ligationvectors. Such vectors will be more universally useful for thedevelopment of cancer vaccines.

SUMMARY OF THE INVENTION

The invention relates to methods for the identification of targetantigens recognized by CTLs, and the formulation and use of suchantigens in immunogenic compositions or vaccines to induce cell-mediatedimmunity against target cells, such as tumor cells, that express thetarget antigens.

Two basic approaches are described for the identification of targetantigens. In one approach, CTLs generated against authentic targetcells, such as tumor cells, in animals tolerized to non-target (e.g.,non-tumorigenic) cellular counterparts are used to screen expressionlibraries made from target cell-derived (e.g., tumor-derived) DNA, RNAor cDNA to identify clones expressing target antigens. The CTLsgenerated by the methods described herein are not cross-reactive withnormal cells, and thus are better tools for screening. Improvedexpression libraries are also described.

In a second approach for identifying target antigens, products of genesdifferentially expressed in target cells, such as tumor cells, are usedto immunize animals to generate HLA-restricted CTLs which are evaluatedfor activity against authentic target cells. Like the first approach,this second strategy could also be particularly useful for identifyingepitopes for many human tumor types where it has not been possible togenerate tumor-specific CTLs directly from patients. In addition, it mayidentify cryptic antigens of the intact tumor cell—i.e., tumor cellproducts which can become immunogenic, if the representation oftumor-specific CTLs is first augmented by vaccination with that tumorcell product. Modified methods for differential display that improveresolution and reduce false positives are described.

In accordance with the present invention, the target cell is a cell towhich it is desirable to induce a cell-mediated immune response.Examples of target cells in the body include, but are not limited to,tumor cells, malignant cells, transformed cells, cells infected with avirus, fungus, or mycobacteria, or cells subject to any other diseasecondition which leads to the production of target antigens.

The invention also encompasses the high yield expression of candidatetarget antigens, and production of recombinant viruses for vaccineformulation.

Abbreviations

-   CTLs—cytotoxic T lymphocytes (T cells)-   PBL—peripheral blood lymphocytes-   RDA—Representational Difference Analysis-   TIL—tumor infiltrating lymphocytes

DESCRIPTION OF THE FIGURES

FIG. 1. Nucleotide Sequence of p7.5/tk (SEQ ID NO:1) and pEL/tk (SEQ IDNO:3). The nucleotide sequence of the promoter and beginning of thethymidine kinase gene for v7.5/tk and vEL/tk.

FIG. 2. Modifications in the nucleotide sequence of the p7.5/tk (SEQ IDNO:5) vaccinia transfer plasmid. Four new vectors, p7.5/ATG0/tk (SEQ IDNO:6), p7.5/ATG1/tk (SEQ ID NO:7), p7.5/ATG2/tk (SEQ ID NO:8) andp7.5/ATG3/tk (SEQ ID NO:9) have been derived as described in the textfrom the p7.5/tk vaccinia transfer plasmid. Each vector includes uniqueBamHI, SmaI, PstI, and SalI sites for cloning DNA inserts that employeither their own endogenous translation initiation site (in vectorp7.5/ATG0/tk (SEQ ID NO:6)) or make use of a vector translationinitiation site in any one of the three possible reading frames(p7.5/ATG1/tk (SEQ ID NO:7), p7.5/ATG2/tk (SEQ ID NO:8) and p7.5/ATG3/tk(SEQ ID NO:9)).

FIG. 3. Schematic of the Clontech PCR SELECT™ method of RepresentationalDifference Analysis. Adapted from information provided by themanufacturer.

FIG. 4. BCA 39 tumor DNA fragments amplified by PCR following RDAsubtraction of B/c.N parental sequences. Nested primers incorporatedinto the RDA adapters ligated to BCA 39 tumor cell cDNA were employedfor sequential PCR amplification of the DNA fragments recovered fromRDA. Bands are resolved on a 2% Metaphor agarose gel. One additional lowmolecular weight band ran off the illustrated gel and was recovered froma shorter electrophoretic run.

FIG. 5. Hybridization of an RDA fragment of (A) an IAP pol gene or (B) afragment of the ubiquitously expressed murine G3PDH cDNA to Northernblots of BCA 39 tumor RNA. 15 micrograms of total RNA was transferredfrom 1% alkaline agarose gel to Genescreen nylon membrane by capillaryblot in 10×SSC. The Northern blot was first hybridized to the ³²Plabeled RDA clone 1 DNA (10⁵ cpm/ml Stark's hybridization buffer), thenstripped and hybridized with a 350 bp fragment of G3PDH cDNA.

FIG. 6. RDA clones encoding fragments of IAP gene elements compared withthe full length IAP clone MIA14. One or both terminal regions (filledrectangular box) of each RDA clone were sequenced to identify homologyto subregions of an IAP element. The extent of overlap was estimatedfrom either the fragment size or, where the sequence at both termini ofa fragment was determined, the known IAP MIA14 sequence spanning the twotermini of that fragment. In one case, clone 2.19, the two measures werenot consistent suggesting a deletion in this IAP fragment in BCA 39tumor cells.

FIG. 7. Modified Differential Display of cDNA of parental cell B/c.N andtumors BCA 39, BCA 34, BCA 22, and BCB 13. Fragments of parental andtumor cell cDNA were amplified with one pair of arbitrary decamers,MR_(—)1 (TAC AAC GAG G) (SEQ ID NO:11) and MR_(—)5 (GGA CCA AGT C)(SEQID NO:13). For each cell line, first strand cDNA synthesis wasseparately primed with MR-1 or MR_(—)5. The two cDNA preparations werethen pooled for PCR amplification with both MR_(—)1 and MR_(—)5. Anumber of bands can be identified that are associated with all fourtumors but not with the immortalized, non-tumorigenic parental cellline.

FIGS. 8A and 8B. Differential expression in tumor lines of differentialdisplay clone 90. RNase protection assay: 300 picograms of clone 90antisense probe was hybridized with 5 micrograms total RNA prior toRNase digestion and analysis of protected fragments on 5% denaturingPAGE.

FIG. 9. Gene isolation in solution. Schematic of a method for selectionof longer length cDNA from single strand circles rescued from a phagemidlibrary. DNA fragments identified through RDA or Modified DifferentialDisplay are employed to select more full length cDNA.

FIG. 10. Restriction Enzyme Analysis of Virus Genomes Using CHEF Gel.BSC-1 cells were infected at high multiplicity of infection (moi) byvaccinia WR, vEL/tk, v7.5/tk, or vNotI/tk. After 24 hours the cells wereharvested and formed into agarose plugs. The plugs were equilibrated inthe appropriate restriction enzyme buffer and 1 mM PMSF for 16 hours atroom temperature, incubated with restriction enzyme buffer, 100 ng/mlBovine Serum Albumin and 50 units NotI or ApaI for two hours at 37° C.(NotI) or room temperature (ApaI) and electrophoresed in a 1.0% agarosegel on a Bio-Rad CHEFII apparatus for 15 hours at 6 V/cm with aswitching time of 15 seconds. The leftmost sample contains lambda DNA,the second sample contains undigested vaccinia DNA, and the remainder ofthe samples contain the DNA samples described above each well digestedwith ApaI or NotI where vEL refers to vEL/tk and v7.5 refers to v7.5/tk.The lower portion of the figure is a schematic map showing the locationof the NotI and ApaI sites in each virus.

FIGS. 11 A and 11B. Southern Blot Analysis of Viral Genomes p7.5/tk(FIG. 11A) and pEUtk (FIG. 11B). The viruses v7.5/tk and vEL/tk wereused to infect a well of a 6 well dish of BSC-1 cells at highmultiplicity of infection (moi) and after 48 hours the cells wereharvested and the DNA was isolated using DNAzoI (Gibco). The final DNAproduct was resuspended in 50 microliters of TE 8.0 and 2.5 microliterswere digested with HindIII, HindIII and ApaI, or HindIII and Not I,electrophoresed through a 1.0% agarose gel, and transferred to Nytran(Schleicher and Schuell) using a Turboblotter (Schleicher and Schuell).The samples were probed with p7.k/tk (FIG. 11A) or pEL/tk (FIG. 11B)labeled with ³²P using Random Primer DNA Labeling Kit (Bio-Rad) inQuickHyb (Stratagene). The lower portion of the figure denotes a map ofthe HindIII J fragment with the positions of the HindIII, NotI, and ApaIsites illustrated. The leftmost 0.5 kilobase fragment haselectrophoresed off the bottom of the gel.

FIG. 12. Analysis of v7.5/tk and vEL/tk by PCR. One well of a 6 welldish of BSC-1 cells was infected with v7.5/tk, vEL/tk, vNotI/tk, vpNotI,vNotI/lacZ/tk, or wild type vaccinia WR at high multiplicity ofinfection (moi) and after 48 hours the cells were harvested, and the DNAwas isolated using DNAzoI (Gibco). The final DNA product was resuspendedin 50 microliters of TE (10 mM TrisHCl, pH8.0. 1 mM EDTA) and used in aPCR with primers MM407 and MM408. The primers are separated by 518nucleotides in vaccinia WR and yield a fragment containing the Nterminus of the thymidine kinase gene. The products were electrophoresedthrough a 2% agarose gel. The leftmost sample contains phiX 174 HaeIIIdigestion products; all others contain the PCR product using primersMM407 and MM408 with the DNA sample indicated above the well

FIG. 13. Promoter strength of recombinant viruses. The units of β-gluactivity were determined as described by Miller (10) as adapted for96-well plates. The A₄₀₅ values were determined on a microplate reader(Dynatech MR3000) and the β-glu activity was determined by comparison toβ-glu (Clontech) standards analyzed in the same assay.

FIG. 14. Plaque assay on vEL/tk. Ten-fold dilutions of vEL/tk wereincubated with Hutk cells (top to bottom) for one hour at 37° C. in 1 mlof E-MEM (Gibco) with 10% Fetal Bovine Serum for one hour, the media wasreplaced with 3 ml of E-MEM with 5% methyl cellulose (Sigma M-0387), 5%Fetal Bovine Serum and HAT supplement (Gibco), 25 or 125 mMbromodeoxyuridine, or no drug, incubated for 48 hours at 37° C., andstained with 0.5% Crystal Violet (Sigma C 0775), 20% ethanol, 7.5%formaldehyde.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for the identification oftarget antigens recognized by CTLs, and the use of such antigens inimmunogenic compositions or vaccines to induce a cell-mediated immuneresponse against cells which express the target antigens.

In one embodiment of the invention, tumor-specific CTLs generated inanimals are used to screen expression libraries generated from tumorcell DNA, RNA or cDNA to identify reactive target antigens. To this end,animals tolerized with a non-tumorigenic human cell line are immunizedwith tumor cells derived from the non-tumorigenic cell line. Theresulting CTLs, which are tumor-specific and not cross-reactive withnormal cells, can be used to screen expression libraries constructedfrom tumor-cell derived DNA, RNA or cDNA. Clones so identified in thelibrary encode target antigens which are candidates for the immunogeniccompositions and vaccines of the invention. Improved and modifiedvaccinia virus vectors for efficient construction of such DNA librariesusing a “trimolecular recombination” approach are described to improvescreening efficiency.

It is a preferred embodiment of the invention to tolerize animals, suchas normal or transgenic mice, with normal human cells prior toimmunizing with human tumor cells. Tolerance induction is preferredbecause the animal's immune response would otherwise be dominated byspecificity for a large number of broadly expressed human proteins thatare not specifically associated with tumor transformation. In aparticularly preferred embodiment, and to enhance the efficiency of thisapproach, it is convenient to work with human tumors that are derivedfrom an immortalized, non-tumorigenic human cell line by in vitrocarcinogenesis or oncogene transformation. This provides a ready sourceof the normal control cells for an extended tolerization protocol inboth neonatal and adult mice. For example, CTLs generated by thisapproach (see Example 2 below) can be employed in a selection procedure(such as that described in Example 3 below) to isolate recombinantclones that encode the target antigens from a tumor cDNA library, forexample, such as that constructed in vaccinia virus by tri-molecularrecombination (see Example 1 below).

In another embodiment of the invention, the products of genes that aredifferentially expressed in tumor cells are used to generateHLA-restricted CTLs which are evaluated for activity against authentictumor cells. It is particularly preferred if methods such asRepresentational Difference Analysis (RDA) and differential display areemployed to identify gene fragments that are differentially expressed intumor versus normal cells. Conveniently, if it is determined that thesegene products are broadly expressed in other related tumors (see, forexample, Examples 5 and 6 below), they may be used to select longerclones from the library (see, for example, Example 4) which may betested for the ability to induce a tumor-specific immune response in,for example, human CD8 and HLA transgenic mice (see, for example,Example 7). Gene products which generate tumor-specific cell mediatedimmunity are also candidates for the immunogenic compositions andvaccines of the invention. Improved methods for differential display aredescribed that enhance screening efficiency by reducing false positives,and enhance the efficiency for isolating full length cDNAs.

The antigens identified using any of the foregoing strategies can beproduced in quantity by recombinant DNA methods and formulated with anadjuvant that promotes a cell-mediated immune response. Preferably, theDNA encoding the target antigen is engineered into a recombinant virusthat can be used to vaccinate animal hosts, including humans. In thisregard, improved direct ligation vaccinia vectors are described that canbe used to generate vaccines.

Another therapeutic strategy of the invention is to design vaccines thattarget a small set of HLA class I molecules which are expressed atelevated frequencies across ethnic populations. Extensivecharacterization of peptide binding motifs of different human class IMHC molecules has suggested that there are four major subtypes of HLA-Aand HLA-B alleles (Sidney, J., et al., Immunol. Today 17:261-266 (1996))such that many peptides will bind to multiple members of a single group.The present invention also provides methods to target vaccines forpatients based on their membership in a class I MHC group. In specificembodiments, class I MHC subtypes A2, A3, B7 and B44 are targeted. Eachgroup has an average representation across ethnic populations of between40% and 50%. It is estimated that the combination of all four groups(which include 50% to 60% of all known HLA-A and HLA-B alleles) covers95% of the human population. In a specific embodiment, HLA-A2.1, themost frequently expressed HLA allele in human populations (Caucasian43%, Black 20%, Chinese 25%) and the dominant member of the A2 subtype,is targeted.

Although the methods of the invention described are used to identifyreactive target antigens in tumor cells, the methods may also be used toidentify target antigens in other target cells against which it isdesirable to induce cell-mediated immunity. For example, thedifferential immunogenicity methods of the invention can be applied toidentify immunogenic molecules of cells infected with virus, fungus ormycobacteria by tolerization of mice with uninfected cells followed byimmunization with infected cells at different times after infection. Theisolated CTLs can be employed to select recombinants that encode targetantigens in a plasmid or viral expression library. An expression librarycan be constructed with cDNA isolated from the infected cell in avaccinia virus vector using tri-molecular recombination.

A particular advantage of this approach is that it will identifypotential antigens expressed not only by the pathogen but also by thehost cell whose gene expression is altered as a result of infection.Since many pathogens elude immune surveillance by frequent reproductionand mutation, it may be of considerable value to develop a vaccine thattargets host gene products that are not likely to be subject tomutation.

The differential gene expression strategies of the present invention mayalso be applied to identify immunogenic molecules of cells infected withvirus, fungus or mycobacteria. More stable and/or previouslyunidentified antigens encoded by genes of either pathogen or host,including those which might remain cryptic without prior specificvaccination, may be identified.

Pathogens include, but are not limited to: viral pathogens, such ashuman immunodeficiency virus, Epstein Barr virus, hepatitis virus,herpes virus, human papillomavirus, cytomegalovirus, respiratorysyncytial virus; fungal pathogens, such as Candida albicans,pneumocystis carnii; and mycobacterial pathogens, such as M.tuberculosis, M. avium.

The following details and examples mention primarily target antigens intumor cells. As will be appreciated from the foregoing, the methods ofthe invention may be adapted to identify target antigens in other targetcells, such as virally infected cells, and may be useful in developingvaccines.

Identifying Target Antigens for Use in Vaccines

The subsections below describe two strategies that can be used toidentify target antigens or epitopes that are candidates for use inimmunogenic formulations or vaccines. The two strategies describedherein may be applied to identify target epitopes which include, but arenot limited to, tumor specific, epitopes specific to a cell infectedwith a virus. fungus or mycobacteria, and/or epitopes specific to anautoimmune disease.

Induction of Cytotoxic T Lymphocytes Specific for Human Tumors and TheirUse to Select DNA Recombinants that Encode Target Epitopes

In this embodiment of the invention, cytotoxic T cells specific forhuman tumors are induced in animals which have been tolerized with anon-tumorigenic, immortalized normal human cell line that does notexpress costimulator activity. These animals are subsequently immunizedwith costimulator transfected (e.g., B7 transfected) tumor cells derivedby in vitro mutagenesis or oncogene transformation from that same normalimmortalized human cell line. An alternative source of matched normaland tumor cell pairs that could be employed in this same fashion is toderive normal and tumor cell lines from different tissue samples of thesame patient. For purposes of immunization, costimulator activity couldalso be introduced in these tumor cells by transfection with murine B7.This immunization regimen gives rise to tumor-specific CTL that are notcrossreactive on the homologous normal cells. The primary purpose ofinducing tumor-specific CTL is that they can be employed, as describedbelow, to select for clones of recombinant tumor DNA that encode thetarget antigen. Such antigens, because they are differentiallyimmunogenic in tumor as compared to normal cells, are candidates forimmunogenic formulations or vaccines. Mammals of different species, mostcommonly diverse strains of inbred mice, can be employed for thispurpose. Whether a particular formulation or vaccine is immunogenic inany particular individual will depend on whether specific peptidesderived from that antigen can be processed and presented in associationwith the particular MHC molecules expressed by that individual. Tonarrow the focus of this selection process to antigens from whichpeptides can be derived that associate with a particular human HLAmolecule, it is possible, as described in Example 2, to derive directlyHLA restricted CTL from HLA and human CD8 transgenic mice.Alternatively, differentially immunogenic molecules of the human tumorcan be initially identified employing tumor-specific CTL restricted toany animal MHC. Antigens so identified can subsequently be characterizedfor the ability to be processed and presented in association withdifferent human HLA types by primary in vitro stimulation of humanperipheral blood lymphocytes (PBL), or, as described in Example 7, byimmunization of HLA and human CD8 transgenic mice. The HLA transgenepermits selection of a high affinity, HLA-restricted T cell repertoirein the mouse thymus. In addition, a human CD8 transgene is mostpreferable because murine CD8 does not interact efficiently with humanclass I MHC.

The method to determine differential immunogenicity can be carried outin normal mice if genes encoding mouse MHC molecules are introduced intothe human cell lines by transfection (Kriegler, M., Gene transfer andexpression: A laboratory manual, W.H. Freeman and Co., N.Y., (1991)).Alternatively, antigens of the human cell lines may be re-presented bymurine professional antigen presenting cells in vivo (Huang, et al.,Science 264:961-965 (1994)) and in vitro (Inaba, et al., J. Exp. Med.176:1702 (1992); Inaba, et al., J. Exp. Med. 178:479-488 (1993)). Toinduce T cell tolerance during re-presentation of human antigens bymurine dendritic cells it may be necessary to block costimulatoractivity with anti-B7.1 and anti-B7.2 antibodies. Specificity of the CTLgenerated in this way may be determined by comparing lysis of humantumor and normal target cells that have been transfected with HLA classI or that have been infected with HLA class I or that have been infectedwith HLA class I recombinant vaccinia virus.

Since immunogenicity of antigen in any individual depends on whetherpeptides derived from the antigen can be presented to T cells inassociation with MHC molecules of that particular individual, it may beseparately determined by immunization of human volunteers or of humanCD8 and HLA transgenic mice, which human HLA molecules are able topresent peptides of any identified antigen. The two issues ofimmunogenicity and HLA associated presentation can be addressedsimultaneously if HLA transgenic mice rather than normal mice areemployed in the initial immunization.

The construction of transgenic mice is well known in the art and isdescribed, for example. in Manipulating the Mouse Embryo: A laboratoryManual, Hogan, et al., Cold Spring Harbor Press, second edition, 1994.Human CD8 transgenic mice may be constructed by the method of LaFace, etal., J. Exp. Med. 182:1315-25 (1995). Construction of new lines oftransgenic mice expressing the human CD8alpha and CD8beta subunits maybe made by insertion of the corresponding human cDNA into a human CD2minigene based vector for T cell-specific expression in transgenic mice(Zhumabekov, et al., J. Immunol. Methods 185:133-140 (1995)). HLA classI transgenic mice may be constructed by the methods of Chamberlain. etal., Proc. Natl. Acad. Sci. USA 85:7690-7694 (1988) or Bernhard, et al.,J. Exp. Med. 168: 1157-62 (1988) or Vitiello, et al., J. Exp. Med. 173:1007-1015 (1991) or Barra, et al., J. Immunol. 150: 3681-9 (1993).

Construction of additional HLA class I transgenic mice may be achievedby construction of an H-2 Kb cassette that includes 2 kb of upstreamregulatory region together with the first two introns previouslyimplicated in gene regulation (Kralova, et al., EMBO J. 11:4591-4600(1992)). Endogenous translational start sites are eliminated from thisregion and restriction sites for insertion of HLA cDNA are introducedinto the third exon followed by a polyA addition site. By including anadditional 3 kb of genomic H-2 Kb sequence at the 3′ end of thisconstruct, the class I gene can be targeted for homologous recombinationat the H-2 Kb locus in embryonic stem cells. This has the advantage thatthe transgene is likely to be expressed at a defined locus known to becompatible with murine class I expression and that these mice are likelyto be deficient for possible competition by H-2 Kb expression at thecell membrane. It is believed that this will give relativelyreproducible expression of diverse human HLA class I cDNA introduced inthe same construct.

Most preferably, the tumor cell lines are a panel of tumor cell linesthat are all derived from a single immortalized, non-tumorigenic cellline. Non-tumorigenic cells are most preferable for inducing toleranceto the large number of normal human proteins that are also expressed intumor cells.

Preferably, screening is performed on such a panel of tumor cell lines,independently derived from the same normal cells by diverse carcinogensor oncogene transformation. Screening of such a panel of tumor celllines makes it possible to filter out antigenic changes that arecarcinogen specific or that may arise by random genetic drift during invitro propagation of a tumor cell line.

The tumor-specific CTLs generated as described above can be used toscreen expression libraries prepared from the target tumor cells inorder to identify clones encoding the target epitope. DNA librariesconstructed in a viral vector infectious for mammalian cells asdescribed herein can be employed for the efficient selection of specificrecombinants by CTLs. Major advantages of these infectious viral vectorsare 1) the ease and efficiency with which recombinants can be introducedand expressed in mammalian cells, and 2) efficient processing andpresentation of recombinant gene products in association with MHCmolecules of the infected cell. At a low multiplicity of infection(m.o.i.), many target cells will express a single recombinant which isamplified within a few hours during the natural course of infection.

In one embodiment of the invention, a representative DNA library isconstructed in vaccinia virus. Preferably, a tri-molecular recombinationmethod employing modified vaccinia virus vectors and related transferplasmids is used to construct the representative DNA library in vacciniavirus. This method generates close to 100% recombinant vaccinia virus(see Example 1).

In a preferred embodiment (see also Example 9), a vaccinia virustransfer plasmid pJ/K, a pUC 13 derived plasmid with a vaccinia virusthymidine kinase gene containing an in-frame Not I site, is furthermodified to incorporate one of two strong vaccinia virus promoters,e.g., either a 7.5K vaccinia virus promoter or a strong syntheticearly/late (E/L) promoter, followed by Not I and Apa I restrictionsites. The Apa I site is preferably preceded by a strong translationalinitiation sequence including the ATG codon. This modification ispreferably introduced within the vaccinia virus thymidine kinase (tk)gene so that it is flanked by regulatory and coding sequences of theviral tk gene. Each of the two modifications within the tk gene of aplasmid vector may be transferred by homologous recombination in theflanking tk sequences into the genome of the Vaccinia Virus WR strainderived vNotI vector to generate two new viral vectors. Importantly,following Not I and Apa I restriction endonuclease digestion of thesetwo viral vectors, two large viral DNA fragments can be isolated eachincluding a separate non-homologous segment of the vaccinia tk gene andtogether comprising all the genes required for assembly of infectiousviral particles.

In one embodiment, such modifications are introduced in the ModifiedVirus Ankara (MVA) strain of vaccinia, which is replication deficient inmammalian cells (Meyer, et al., J. Gen. Virol. 72:1031-1038 (1991)).

In a preferred embodiment, the following method is used to enrich for,and select for those cells infected with the recombinant viruses thatexpress the target epitopes of specific cytotoxic T cells. An adherentmonolayer of cells is infected with a recombinant viral library, e.g. avaccinia recombinant viral library, at m.o.i. less than or equal to 1.It is important that these cells do not themselves express the targetepitopes recognized by specific CTLs but that these epitopes arerepresented in the viral library. In addition, for selection by CTLs,the infected cells must express an appropriate MHC molecule that canassociate with and present the target peptide to T cells.

After 12 hours infection with recombinant virus, the monolayer is washedto remove any non-adherent cells. CTLs of defined specificity are addedfor 30 min. During this time, some of the adherent cells infected with arecombinant particle that leads to expression of the target epitope willinteract with a specific CTL and undergo a lytic event. Cells thatundergo a lytic event are released from the monolayer and can beharvested in the floating cell population. The above-described protocolis repeated for preferably five or more cycles, to increase the level ofenrichment obtained by this procedure.

Screening Cytotoxic Lymphocytes Generated Against Products of GenesDifferentially Expressed in Tumor Cells for Activity Against AuthenticTumor Cells

In this embodiment of the invention, the products of genes that aredifferentially expressed in a tumor are used to generate HLA-restrictedCTLs (e.g., by immunization of transgenic animals or in vitrostimulation of human PBL with antigen presenting cells that express theappropriate MHC ). The CTLs so generated are assayed for activityagainst authentic tumor cells in order to identify the differentiallyexpressed gene which encodes the effective target epitope.

In essence, this approach to identify tumor-specific antigens is thereverse of the strategy described in the preceding section. Rather thanisolating CTLs generated against authentic tumor cells to screenexpression libraries of tumor-specific cDNA, the tumor-specific cDNA orgene products (i.e., the product of genes differentially expressed intumors) are used to generate CTLs which are then screened usingauthentic tumor. This strategy is quite advantageously used to identifytarget epitopes for many human tumor types where it has not beenpossible to generate tumor-specific CTL directly from patients. Thisstrategy provides an additional advantage in that cryptic tumor antigenscan be identified. Rather than only assaying for what is immunogenic ona tumor cell, this embodiment of the invention allows for the evaluationand assessment of tumor cell products that can become immunogenic if therepresentation of tumor-specific T cells is first augmented byvaccination.

Differentially expressed genes derived from the tumor can be identifiedusing standard techniques well known to those skilled in the art (e.g.,see Liang & Pardee, Science 257:967-971 (1992)), which is incorporatedby reference herein in its entirety). Preferably, the improveddifferential display methods described in Example 4, infra, may be usedto reduce false positives and enhance the efficiency for isolating fulllength cDNAs corresponding to the identified DNA fragments. Eachdifferentially expressed gene product is potentially immunogenic, andmay be represented as a low-abundance or high abundance transcript.

In order to identify the differentially expressed gene products thatmight be candidates for tumor immunotherapy, it is necessary to have ameans of delivering the product for immunization in an environment inwhich T cell responses to peptides associated with human HLA can beinduced. To this end, the differentially expressed cDNA is incorporatedinto an expression vector, preferably a viral vector (such as thevaccinia vectors described herein) so that quantities of the geneproduct adequate for immunization are produced. Immunization can beaccomplished using the recombinantly expressed gene product formulatedin a subunit vaccine (e.g., mixed with a suitable adjuvant that canpromote a cell mediated immune response). Preferably a recombinant viralexpression vector, such as vaccinia. can be used to immunize (Bennock &Yewdell, Current Topics In Microbiol. and Immunol. 163:153-178 (1990)).Most preferably, transgenic mice are employed which express a humanclass I MHC molecule, so that HLA-restricted murine cytotoxic T cellsspecific for the gene product can be induced and isolated (Shirai, M.,et al., J. Immunol. 154:2733-42 (1995); Wentworth et al., Eur. J. ofImmunol. 26:97-101(1996)). Alternatively, human PBL are stimulated invitro with antigen presenting cells that express homologous HLA.

The significance of HLA compatibility is that T cells recognize peptidesthat bind to, and are transported to the surface of antigen presentingcells in association with major histocompatibility molecules. T cells ofHLA transgenic mice are, therefore, primed to recognize a specificpeptide in association with the expressed human HLA and crossreactivitywith human tumor cells depends on expression of that same tumor peptidein association with the same HLA molecule.

The CTLs induced by the immunization can be tested for cross reactivityon HLA compatible tumors that express the corresponding mRNA. The CTLscan be assayed for their ability to kill authentic tumor cells in vitroor in vivo. To this end, assays described in Example 2 can be used, orother similar assays for determining tumor cell specificity and killingwhich are well known to those skilled in the art.

Using this approach, target epitopes which are particularly goodcandidates for tumor immunotherapy in human patients are identified asthose which meet the following criteria: (a) the gene is differentiallyexpressed in multiple human tumors; (b) the gene products areimmunogenic in association with HLA; and (c) the specific CTLs inducedare cross reactive on human tumor cells.

Vaccine Formulations

The present invention encompasses the expression of the identifiedtarget epitope in either eucaryotic or procaryotic recombinantexpression vectors; and the formulation of the identified epitope asimmunogenic and/or antigenic compositions. In accordance with thepresent invention, the recombinantly expressed target epitope may beexpressed, purified and formulated as a subunit vaccine. The identifiedtarget epitope may also be constructed into viral vectors for use invaccines. In this regard, either a live recombinant viral vaccine, aninactivated recombinant viral vaccine, or a killed recombinant viralvaccine can be formulated.

Expression of the Target Epitope in Procaryotic and EucaryoticExpression Systems

The present invention encompasses expression systems, both eucaryoticand procaryotic expression vectors, which may be used to express theidentified target epitope. The identified epitope may be expressed inboth truncated or full-length forms of the epitope, in particular forthe formation of subunit vaccines.

The present invention encompasses the expression of nucleotide sequencesencoding the identified epitopes and immunologically equivalentfragments. Such immunologically equivalent fragments may be identifiedby making analogs of the nucleotide sequence encoding the identifiedepitopes that are truncated at the 5′ and/or 3′ ends of the sequenceand/or have one or more internal deletions, expressing the analognucleotide sequences, and determining whether the resulting fragmentsimmunologically are recognized by the epitope specific CTLs and induce acell-mediated immune response.

The invention encompasses the DNA expression vectors that contain any ofthe foregoing coding sequences operatively associated with a regulatoryelement that directs expression of the coding sequences and geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

The target epitope gene products or peptide fragments thereof, may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing the epitope gene polypeptides andpeptides of the invention by expressing nucleic acid containing epitopegene sequences are described herein. Methods which are well known tothose skilled in the art can be used to construct expression vectorscontaining epitope gene product coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, supra, and Ausubel etal., 1989, supra. Alternatively, RNA capable of encoding glycoproteinepitope gene product sequences may be chemically synthesized using, forexample, synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety.

The invention also encompasses nucleotide sequences that encode peptidefragments of the identified epitope gene products. For example,polypeptides or peptides corresponding to the extracellular domain ofthe selected epitope may be useful as “soluble” protein which wouldfacilitate secretion, particularly useful in the production of subunitvaccines. The selected epitope gene product or peptide fragmentsthereof, can be linked to a heterologous epitope that is recognized by acommercially available antibody is also included in the invention. Adurable fusion protein may also be engineered; i.e., a fusion proteinwhich has a cleavage site located between the selected epitope sequenceand the heterologous protein sequence, so that the selected epitope canbe cleaved away from the heterologous moiety. For example, acollagenasecleavage recognition consensus sequence may be engineered between theselected epitope protein or peptide and the heterologous peptide orprotein. The epitopic domain can be released from this fusion protein bytreatment with collagenase. In a preferred embodiment of the invention,a fusion protein of glutathione-S-transferase and the selected epitopeprotein may be engineered.

The selected epitope proteins of the present invention for use invaccine preparations, in particular subunit vaccine preparations, aresubstantially pure or homogeneous. The protein is consideredsubstantially pure or homogeneous when at least 60 to 75% of the sampleexhibits a single polypeptide sequence. A substantially pure proteinwill preferably comprise 60 to 90% of a protein sample, more preferablyabout 95% and most preferably 99%. Methods which are well known to thoseskilled in the art can be used to determine protein purity orhomogeneity, such as polyacrylamide gel electrophoresis of a sample,followed by visualizing a single polypeptide band on a staining gel.Higher resolution may be determined using HPLC or other similar methodswell known in the art.

The present invention encompasses polypeptides which are typicallypurified from host cells expressing recombinant nucleotide sequencesencoding these proteins. Such protein purification can be accomplishedby a variety of methods well known in the art. In a preferredembodiment, the epitope protein of the present invention is expressed asa fusion protein with glutathione-S-transferase. The resultingrecombinant fusion proteins purified by affinity chromatography and theepitope protein domain is cleaved away from the heterologous moietyresulting in a substantially pure protein sample. Other methods known tothose skilled in the art may be used; see for example, the techniquesdescribed in “Methods In Enzymology”, 1990, Academic Press, Inc., SanDiego, “Protein Purification: Principles and Practice”, 1982,Springer-Verlag, New York, which are incorporated by reference herein intheir entirety.

Eucaryotic and Procaryotic Expression Vectors

The present invention encompasses expression systems, both eucaryoticand procaryotic expression vectors, which may be used to express theselected epitope. A variety of host-expression vector systems may beutilized to express the selected target epitope gene of the invention.Such host-expression systems represent vehicles by which the codingsequences of interest may be produced and subsequently purified, butalso represent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, exhibit the selected epitopegene product of the invention in situ. These include but are not limitedto microorganisms such as bacteria (e.g., E. coli, B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing selected epitope gene product codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the selected epitopegene product coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing theselected epitope gene product coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingselected epitope gene product coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

Host Cells

The present invention encompasses the expression of the selected epitopein animal and insect cell lines. In a preferred embodiment of thepresent invention, the selected epitope is expressed in a baculovirusvector in an insect cell line to produce an unglycosylated antigen. Inanother preferred embodiment of the invention, the selected epitope isexpressed in a stably transfected mammalian host cell, e.g., Tlymphocyte cell line to produce a glycosylated antigen. The selectedepitopes which are expressed recombinantly by these cell lines may beformulated as subunit vaccines.

A host cell strain may be chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. Such modifications (e.g., glycosylation) andprocessing (e.g. cleavage) of protein products may be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification of theforeign protein expressed. To this end, eucaryotic host cells whichpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product maybe used. Such mammalian host cells include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and W138 cell lines.

For long term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe selected target epitope may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines. This method mayadvantageously be used to engineer cell lines which express the selectedepitope gene products. Such cell lines would be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the selected epitope gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223(1977)), hypoxanthine-guanine phlosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes canbe employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., Natl. Acad. Sci. USA 77:3567 (1980); O'Hare, et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981)); and hygro, whichconfers resistance to hygromycin (Santerre, et al., Gene 30:147 (1984)).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht. et al., Proc. Natl. Acad Sci. USA 88: 8972-8976(1991)). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁻nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

Expression of Target Epitope in Recombinant Viral Vaccines

In another embodiment of the present invention, either a liverecombinant viral vaccine or an inactivated recombinant viral vaccineexpressing the selected target epitope can be engineered. A live vaccinemay be preferred because multiplication in the host leads to a prolongedstimulus of similar kind and magnitude to that occurring in naturalinfections, and therefore, confers substantial, long-lasting immunity.Production of such live recombinant virus vaccine formulations may beaccomplished using conventional methods involving propagation of thevirus in cell culture or in the allantois of the chick embryo followedby purification.

In this regard, a variety of viruses may be genetically engineered toexpress the selected epitope. For vaccine purposes, it may be requiredthat the recombinant viruses display attenuation characteristics.Current live virus vaccine candidates for use in humans are either coldadapted, temperature sensitive, or attenuated. The introduction ofappropriate mutations (e.g., deletions) into the templates used fortransfection may provide the novel viruses with attenuationcharacteristics. For example, specific multiple missense mutations thatare associated with temperature sensitivity or cold adaptation can bemade into deletion mutations and/or multiple mutations can be introducedinto individual viral genes. These mutants should be more stable thanthe cold or temperature sensitive mutants containing single pointmutations and reversion frequencies should be extremely low.Alternatively, recombinant viruses with “suicide” characteristics may beconstructed. Such viruses go through only one or a few rounds ofreplication in the host.

For purposes of the invention, any virus may be used in accordance withthe present invention which: (a) displays an attenuated phenotype or maybe engineered to display attenuated characteristics; (b) displays atropism for mammals, in particular humans, or may be engineered todisplay such a tropism; and (c) may be engineered to express theselected target epitope of the present invention.

Vaccinia viral vectors may be used in accordance with the presentinvention, as large fragments of DNA are easily cloned into its genomeand recombinant attenuated vaccinia variants have been described (Meyer,et al., J. Gen. Virol. 72:1031-1038 (1991)). Orthomyxoviruses, includinginfluenza; Paramyxoviruses, including respiratory syncytial virus andSendai virus; and Rhabdoviruses may be engineered to express mutationswhich result in attenuated phenotypes (see U.S. Pat. No. 5,578,473,issued Nov. 26, 1996). These viral genomes may also be engineered toexpress foreign nucleotide sequences, such as the selected epitopes ofthe present invention (see U.S. Pat. No. 5,166,057, issued Nov. 24,1992, incorporated herein by reference in its entirety). Reverse genetictechniques can be applied to manipulate negative and positive strand RNAviral genomes to introduce mutations which result in attenuatedphenotypes, as demonstrated in influenza virus, Herpes Simplex virus,cytomegalovirus and Epstein-Barr virus, Sindbis virus and poliovirus(see Palese et al., Proc. Natl. Acad. Sci. USA 93:11354-11358 (1996)).These techniques may also be utilized to introduce foreign DNA, i.e.,the selected target epitopes, to create recombinant viral vectors to beused as vaccines in accordance with the present invention. In addition,attenuated adenoviruses and retroviruses may be engineered to expressthe target epitope. Therefore, a wide variety of viruses may beengineered to design the vaccines of the present invention, however, byway of example, and not by limitation, recombinant attenuated vacciniavectors expressing the selected target epitope for use as vaccines aredescribed herein.

In one embodiment, a recombinant modified vaccinia variant, ModifiedVirus Ankara (MVA) is used in a vaccine formulation. This modified virushas been passaged for 500 cycles in avian cells and is unable to undergoa full infectious cycle in mammalian cells (Meyer, et al., J. Gen.Virol. 72:1031-1038 (1991)). When used as a vaccine, the recombinantvirus goes through a single replication cycle and induces a sufficientlevel of immune response but does not go further in the human host andcause disease. Recombinant viruses lacking one or more of essentialvaccinia virus genes are not able to undergo successive rounds ofreplication. Such defective viruses can be produced by co-transfectingvaccinia vectors lacking a specific gene(s) required for viralreplication into cell lines which permanently express this gene(s).Viruses lacking an essential gene(s) will be replicated in these celllines but when administered to the human host will not be able tocomplete a round of replication. Such preparations may transcribe andtranslate—in this abortive cycle—a sufficient number of genes to inducean immune response.

Alternatively, larger quantities of the strains can be administered, sothat these preparations serve as inactivated (killed) virus, vaccines.For inactivated vaccines, it is preferred that the heterologous geneproduct be expressed as a viral component, so that the gene product isassociated with the virion. The advantage of such preparations is thatthey contain native proteins and do not undergo inactivation bytreatment with formalin or other agents used in the manufacturing ofkilled virus vaccines.

In another embodiment of the invention, inactivated vaccine formulationsare prepared using conventional techniques to “kill” the recombinantviruses. Inactivated vaccines are “dead” in the sense that theirinfectivity has been destroyed. Ideally, the infectivity of the virus isdestroyed without affecting immunogenicity. In order to prepareinactivated vaccines, the recombinant virus may be grown in cell cultureor in the allantois of the chick embryo, purified by zonalultracentrifugation, inactivated by formaldehyde or β-propiolactone, andpooled. The resulting vaccine is usually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum.

Methods of Treatment and/or Vaccination

Since the identified target epitopes of the present invention can beproduced in large amounts, the antigen thus produced and purified hasuse in vaccine preparations. The target epitope may be formulated into asubunit vaccine preparation, or may be engineered into viral vectors andformulated into vaccine preparations. Alternatively, the DNA encodingthe target epitope may be administered directly as a vaccineformulation. The “naked” plasmid DNA once administered to a subjectinvades cells, is expressed on the surface of the invaded cell andelicits a cellular immune response, so that T lymphocytes will attackcells displaying the selected epitope. The selected epitope also hasutility in diagnostics, e.g., to detect or measure in a sample of bodyfluid from a subject the presence of tumors and thus to diagnose cancerand tumors and/or to monitor the cellular immune response of the subjectsubsequent to vaccination.

The recombinant viruses of the invention can be used to treattumor-bearing mammals, including humans, to generate an immune responseagainst the tumor cells. The generation of an adequate and appropriateimmune response leads to tumor regression in vivo. Such “vaccines” canbe used either alone or in combination with other therapeutic regimens,including but not limited to chemotherapy, radiation therapy, surgery,bone marrow transplantation, etc. for the treatment of tumors. Forexample, surgical or radiation techniques could be used to debulk thetumor mass, after which, the vaccine formulations of the invention canbe administered to ensure the regression and prevent the progression ofremaining tumor masses or micrometastases in the body. Alternatively,administration of the “vaccine” can precede such surgical, radiation orchemotherapeutic treatment.

Alternatively, the recombinant viruses of the invention can be used toimmunize or “vaccinate” tumor-free subjects to prevent tumor formation.With the advent of genetic testing, it is now possible to predict asubject's predisposition for cancers. Such subjects, therefore, may beimmunized using a recombinant vaccinia virus expressing an appropriatetumor-associated antigen.

The immunopotency of the epitope vaccine formulations antigen can bedetermined by monitoring the immune response in test animals followingimmunization or by use of any immunoassay known in the art. Generationof a cell-mediated immune response may be taken as an indication of animmune response. Test animals may include mice, hamsters, dogs, cats,monkeys, rabbits, chimpanzees, etc., and eventually human subjects.

Suitable preparations of such vaccines include injectables, either asliquid solutions or suspensions; sol id forms suitable for solution in,suspension in, liquid prior to injection, may also be prepared. Thepreparation may also be emulsified, or the polypeptides encapsulated inliposomes, the active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine preparation may also include minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and/or adjuvants which enhance the effectiveness ofthe vaccine.

Examples of adjuvants which may be effective, include, but are notlimited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,GM-CSF, QS-21 (investigational drug, Progenics Pharmaceuticals. Inc.),DETOX (investigational drug, Ribi Pharmaceuticals), and BCG.

The effectiveness of an adjuvant may be determined by measuring theinduction of the cellular immune response directed against the targetepitope.

The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from recombinant viruses that direct theexpression of more than one antigen.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc.

Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thecomposition is administered by injection, an ampoule of sterile diluentcan be provided so that the ingredients may be mixed prior toadministration.

In a specific embodiment, a lyophilized epitope of the invention isprovided in a first container; a second container comprises diluentconsisting of an aqueous solution of 50% glycerin, 0.25% phenol, and anantiseptic (e.g., 0.005% brilliant green).

Use of purified antigens as vaccine preparations can be carried out bystandard methods. For example, the purified protein(s) should beadjusted to an appropriate concentration, formulated with any suitablevaccine adjuvant and packaged for use. Suitable adjuvants may include,but are not limited to: mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols; polyanions;peptides; oil emulsions; alum, and MDP. The immunogen may also beincorporated into liposomes, or conjugated to polysaccharides and/orother polymers for use in a vaccine formulation. In instances where therecombinant antigen is a hapten, i.e., a molecule that is antigenic inthat it can react selectively with cognate antibodies, but notimmunogenic in that it cannot elicit an immune response, the hapten maybe covalently bound to a carrier or immunogenic molecule; for instance,a large protein such as serum albumin will confer immunogenicity to thehapten coupled to it. The hapten-carrier may be formulated for use as avaccine.

Many methods may be used to introduce the vaccine formulations describedabove into a patient. These include, but are not limited to, oral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, transdermal, epidural, pulmonary, gastric, intestinal,rectal, vaginal, or urethral routes. When the method of treatment uses alive recombinant vaccinia vaccine formulation of the invention, it maybe preferable to introduce the formulation via the natural route ofinfection of the vaccinia virus, i.e., through a mucosal membrane orsurface, such as an oral, nasal, gastric, intestinal, rectal, vaginal orurethral route. To induce a CTL response, the mucosal route ofadministration may be through an oral or nasal membrane. Alternatively,an intramuscular or intraperitoneal route of administration may be used.Preferably, a dose of 10⁶⁻¹⁰ ⁷ PFU (plaque forming units) of coldadapted recombinant vaccinia virus is given to a human patient.

The precise dose of vaccine preparation to be employed in theformulation will also depend on the route of administration, and thenature of the patient, and should be decided according to the judgmentof the practitioner and each patient's circumstances according tostandard clinical techniques. An effective immunizing amount is thatamount sufficient to produce an immune response to the antigen in thehost to which the vaccine preparation is administered.

Where subsequent or booster doses are required, a modified vacciniavirus such as MVA can be selected as the parental virus used to generatethe recombinant. Alternatively, another virus, e.g., adenovirus, canarypox virus, or a subunit preparation can be used to boost. Immunizationand/or cancer immunotherapy may be accomplished using a combinedimmunization regimen, e.g., immunization with a recombinant vacciniaviral vaccine of the invention and a boost of a recombinant vacciniaviral vaccine. In such an embodiment, a strong secondary CD8 T cellresponse is induced after priming and boosting with different virusesexpressing the same epitope (for such methods of immunization andboosting, see. e.g., Murata et al., Cellular Immunol. 173:96-107). Forexample, a patient is first primed with a vaccine formulation of theinvention comprising a recombinant vaccinia virus expressing an epitope,e.g., a selected tumor-associated antigen or fragment thereof. Thepatient is then boosted, e.g., 21 days later, with a vaccine formulationcomprising a recombinant virus other than vaccinia expressing the sameepitope. Such priming followed by boosting induces a strong secondaryCD8⁺ T cell response. Such a priming and boosting immunization regimenis preferably used to treat a patient with a tumor, metastasis orneoplastic growth expressing the selected tumor-associated antigen.

In yet another embodiment. the recombinant vaccinia viruses can be usedas a booster immunization subsequent to a primary immunization withinactivated tumor cells, a subunit vaccine containing thetumor-associated antigen or its epitope, or another recombinant viralvaccine, e.g., adenovirus, canary pox virus, or MVA.

In an alternate embodiment, recombinant vaccinia virus encoding aparticular tumor-associated antigen, epitope or fragment thereof may beused in adoptive immunotherapeutic methods for the activation of Tlymphocytes that are histocompatible with the patient and specific forthe tumor-associated antigen (for methods of adoptive immunotherapy,see, e.g., Rosenberg, U.S. Pat. No. 4,690,915, issued Sep. 1, 1987;Zarling, et al., U.S. Pat. No. 5,081,029, issued Jan. 14, 1992). Such Tlymphocytes may be isolated from the patient or a histocompatible donor.The T lymphocytes are activated in vitro by exposure to the recombinantvaccinia virus of the invention. Activated T lymphocytes are expandedand inoculated into the patient in order to transfer T cell immunitydirected against the tumor-associated antigen epitope.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers comprising one or more of the ingredients of thevaccine formulations of the invention. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

The invention will be better understood by reference to the specificembodiments detailed in the examples which follow.

EXAMPLE 1 Trimolecular Recombination Employing Modified Vaccinia VirusVectors to Make Expression Libraries

This example describes a tri-molecular recombination method employingmodified vaccinia virus vectors and related transfer plasmids thatgenerates close to 100% recombinant vaccinia virus and, for the firsttime, allows efficient construction of a representative DNA library invaccinia virus.

Construction of the Vectors

The previously described vaccinia virus transfer plasmid pJ/K, a pUC 13derived plasmid with a vaccinia virus thymidine kinase gene containingan in-frame Not I site (Merchlinsky, M. et al., Virology 190:522-526),was further modified to incorporate a strong vaccinia virus promoterfollowed by Not I and Apa I restriction sites. Two different vectors,p7.5/tk and pEL/tk, included, respectively, either the 7.5K vacciniavirus promoter or a strong synthetic early/late (E/L) promoter (FIG. 1).The Apa I site was preceded by a strong translational initiationsequence including the ATG codon. This modification was introducedwithin the vaccinia virus thymidine kinase (tk) gene so that it wasflanked by regulatory and coding sequences of the viral tk gene. Themodifications within the tk gene of these two new plasmid vectors weretransferred by homologous recombination in the flanking tk sequencesinto the genome of the Vaccinia Virus WR strain derived vNotI⁻ vector togenerate new viral vectors v7.5/tk and vEL/tk. Importantly, followingNot I and Apa I restriction endonuclease digestion of these viralvectors, two large viral DNA fragments were isolated each including aseparate non-homologous segment of the vaccinia tk gene and togethercomprising all the genes required for assembly of infectious viralparticles. Further details regarding the construction andcharacterization of these vectors and their alternative use for directligation of DNA fragments in vaccinia virus are described in Example 9infra.

Generation of an Increased Frequency of Vaccinia Virus Recombinants

Standard methods for generation of recombinants in vaccinia virusexploit homologous recombination between a recombinant vaccinia transferplasmid and the viral genome. Table 1 shows the results of a modelexperiment in which the frequency of homologous recombination followingtransfection of a recombinant transfer plasmid into vaccinia virusinfected cells was assayed under standard conditions. To facilitatefunctional assays, a minigene encoding the immunodominant 257-264peptide epitope of ovalbumin in association with H-2K^(b) was insertedat the Not 1 site in the transfer plasmid tk gene. As a result ofhomologous recombination, the disrupted tk gene is substituted for thewild type viral tk+ gene in any recombinant virus. This serves as amarker for recombination since tk− human 143B cells infected with tk−virus are, in contrast to cells infected with wild type tk+ virus,resistant to the toxic effect of BrdU. Recombinant virus can be scoredby the viral pfu on 143B cells cultured in the presence of 125 mM BrdU.

The frequency of recombinants derived in this fashion is of the order of0.1% (Table 1).

TABLE 1 Generation of Recombinant Vaccinia Virus by Standard HomologousRecombination Titer without Titer with % Virus* DNA BrdU BrdURecombinant** vaccinia — 4.6 × 10⁷ 3.0 × 10³ 0.006 vaccinia  30 ngpE/Lova 3.7 × 10⁷ 3.2 × 10⁴ 0.086 vaccinia 300 ng pE/Lova 2.7 × 10⁷ 1.5× 10⁴ 0.056 *vaccinia virus strain vNotl **% Recombinant = (Titer withBrdU/Titer without BrdU) × 100

This recombination frequency is too low to permit efficient constructionof a cDNA library in a vaccinia vector. The following two procedureswere used to generate an increased frequency of vaccinia virusrecombinants.

(i) One factor limiting the frequency of viral recombinants generated byhomologous recombination following transfection of a plasmid transfervector into vaccinia virus infected cells is that viral infection ishighly efficient whereas plasmid DNA transfection is relativelyinefficient. As a result many infected cells do not take up recombinantplasmids and are, therefore, capable of producing only wild type virus.In order to reduce this dilution of recombinant efficiency, a mixture ofnaked viral DNA and recombinant plasmid DNA was transfected into FowlPox Virus (FPV) infected mammalian cells. As previously described byothers (Scheiflinger, F., et al., 1992, Proc. Natl. Acad. Sci. USA89:9977-9981), FPV does not replicate in mammalian cells but providesnecessary helper functions required for packaging mature vaccinia virusparticles in cells transfected with non-infectious naked vaccinia DNA.This modification of the homologous recombination technique aloneincreased the frequency of viral recombinants approximately 35 fold to3.5% (Table 2).

TABLE 2 Generation of Recombinant Vaccinia Virus by Modified HomologousRecombination Titer without with % Virus DNA BrdU BrdU Recombinant* FPVNone 0 0 0 None vaccinia WR 0 0 0 FPV vaccinia WR 8.9 × 10⁶ 2.0 × 10²0.002 FPV vaccinia WR + 5:3 × 10⁶ 1.2 × 10⁵ 2.264 pE/Lova (1:1) FPVvaccinia WR + 8.4 × 10⁵ 3.0 × 10⁴ 3.571 pE/Lova (1:10)

Table 2: Confluent monolayers of BSC1 cells (5×10⁵ cells/well) wereinfected with moi=1.0 of fowlpox virus strain HP1. Two hours latersupernatant was removed, cells were washed 2× with Opti-Mem 1 media, andtransfected using lipofectamine with 600 ng vaccinia strain WR genomicDNA either alone, or with 1:1 or 1:10 (vaccinia:plasmid) molar ratios ofplasmid pE/Lova. This plasmid contains a fragment of the ovalbumin cDNA.which encodes the SIINFEKL epitope, known to bind with high affinity tothe mouse class I MHC molecule K^(b). Expression of this minigene iscontrolled by a strong, synthetic Early/Late vaccinia promoter. Thisinsert is flanked by vaccinia tk DNA. Three days later cells wereharvested, and virus extracted by three cycles of freeze/thaw in dry iceisopropanol/37° C. water bath. Crude virus stocks were titered by plaqueassay on human TK-143B cells with and without BrdU.% Recombinant=(Titer with BrdU/Titer without BrdU)×100

(ii) A further significant increase in the frequency of viralrecombinants was obtained by transfection of FPV infected cells with amixture of recombinant plasmids and the two large approximately 80kilobases and 100 kilobases fragments of vaccinia virus v7.5/tk DNAproduced by digestion with Not I and Apa I restriction endonucleases.Because the Not I and Apa I sites have been introduced into the tk gene,each of these large vaccinia DNA arms includes a fragment of the tkgene. Since there is no homology between the two tk gene fragments, theonly way the two vaccinia arms can be linked is by bridging through thehomologous tk sequences that flank the inserts in the recombinanttransfer plasmid. The results in Table 3 show that >99% of infectiousvaccinia virus produced in triply transfected cells is recombinant for aDNA insert as determined by BrdU resistance of infected tk− cells.

TABLE 3 Generation of 100% Recombinant Vaccinia Virus usingTri-Molecular Recombination Titer without Titer with % Virus DNA BrdUBrdU Recombinant* FPV Uncut 2.5 × 10⁶ 6.0 × 10³ 0.24 v7.5/tk FPVNotl/Apal 2.0 × 10² 0 0 v7.5/tk arms FPV Notl/Apal    6.8 × 10⁴ + 7.4 ×10⁴ 100 v7.5/tk arms 1:1 pE/Lova

Table 3: Genomic DNA from vaccinia strain V7.5/tk (1.2 micrograms) wasdigested with ApaI and NotI restriction endonucleases. The digested DNAwas divided in half. One of the pools was mixed with a 1:1(vaccinia:plasmid) molar ratio of pE/Lova. This plasmid contains afragment of the ovalbumin cDNA, which encodes the SIINFEKL (SEQ ID NO:10) epitope, known to bind with high affinity to the mouse class I MHCmolecule K^(b). Expression of this minigene is controlled by a strong,synthetic Early/Late vaccinia promoter. This insert is flanked byvaccinia tk DNA. DNA was transfected using lipofectamine into confluentmonolayers (5×10⁵ cells/well) of BSC1 cells, which had been infected 2hours previously with moi=1.0 FPV. One sample was transfected with 600ng untreated genomic V7.5/tk DNA. Three days later cells were harvested,and the virus was extracted by three cycles of freeze/thaw in dry iceisopropanol/37° C. water bath. Crude viral stocks were plaqued on TK-143B cells with and without BrdU selection.% Recombinant=(Titer with BrdU/Titer without BrdU)×100Construction of a Representative cDNA Library in Vaccinia Virus

A cDNA library is constructed in the vaccinia vector to demonstraterepresentative expression of known cellular mRNA sequences.

Additional modifications have been introduced into the p7.5/tk transferplasmid and v7.5/tk viral vector to enhance the efficiency ofrecombinant expression in infected cells. These include introduction oftranslation initiation sites in three different reading frames and ofboth translational and transcriptional stop signals as well asadditional restriction sites for DNA insertion.

First, the HindIII J fragment (vaccinia tk gene) of p7.5/tk wassubcloned from this plasmid into the HindIII site of pBS phagemid(Stratagene) creating pBS.Vtk.

Second, a portion of the original multiple cloning site of pBS.Vtk wasremoved by digesting the plasmid with SmaI and PstI, treating with MungBean Nuclease, and ligating back to itself, generating pBS.Vtk.MCS-.This treatment removed the unique SmaI, BamHI, SalI, and PstI sites frompBS.Vtk.

Third, the object at this point was to introduce a new multiple cloningsite downstream of the 7.5k promoter in pBS.Vtk.MCS-. The new multiplecloning site was generated by PCR using 4 different upstream primers,and a common downstream primer. Together, these 4 PCR products wouldcontain either no ATG start codon, or an ATG start codon in each of thethree possible reading frames. In addition, each PCR product contains atits 3 prime end, translation stop codons in all three reading frames,and a vaccinia virus transcription double stop signal. These 4 PCRproducts were ligated separately into the NotI/ApaI sites ofpBS.Vtk.MCS-, generating the 4 vectors, p7.5/ATG0/tk, p7.5/ATG1/tk,p7.5/ATG2/tk, and p7.5/ATG3/tk whose sequence modifications relative tothe p7.5/tk vector are shown in FIG. 2. Each vector includes uniqueBamHI, SmaI, PstI, and SalI sites for cloning DNA inserts that employeither their own endogenous translation initiation site (in vectorp7.5/ATG0/tk) or make use of a vector translation initiation site in anyone of the three possible reading frames (p7.5/ATG1/tk, p7.5/ATG2/tk,and p7.5/ATG3/tk).

In a model experiment cDNA was synthesized from poly-A+ mRNA of a murinetumor cell line (BCA39) and ligated into each of the four modifiedp7.5/tk transfer plasmids. Twenty micrograms of Not I and Apa I digestedv/tk vaccinia virus DNA arms an equal was transfected together with anequimolar mixture of the four recombinant plasmid cDNA libraries intoFPV helper virus infected BSC-1 cells for tri-molecular recombination.The virus harvested had a total titer of 6×10⁶ pfu of which greater than90% were BrdU resistant.

In order to characterize the size distribution of cDNA inserts in therecombinant vaccinia library, individual isolated plaques were pickedusing a sterile pasteur pipette and transferred to 1.5 ml tubescontaining 100 μl Phosphate Buffered Saline (PBS). Virus was releasedfrom the cells by three cycles of freeze/thaw in dry ice/isopropanol andin a 37° C. water bath. Approximately one third of each virus plaque wasused-to infect one well of a 12 well plate containing tk− human 143Bcells in 250 μl final volume. At the end of the two hour infectionperiod each well was overlayed with 1 ml DMEM with 2.5% fetal bovineserum (DMEM-2.5) and with BUdR sufficient to bring the finalconcentration to 125 μg/ml. Cells were incubated in a CO₂ incubator at37° C. for three days. On the third day the cells were harvested,pelleted by centrifugation, and resuspended in 500 μl PBS. Virus wasreleased from the cells by three cycles of freeze/thaw as describedabove. Twenty percent of each virus stock was used to infect a confluentmonolayer of BSC-1 cells in a 50 mm tissue culture dish in a finalvolume of 3 ml DMEM-2.5. At the end of the two hour infection period thecells were overlayed with 3 ml of DMEM-2.5. Cells were incubated in aCO₂ incubator at 37° C. for three days. On the third day the cells wereharvested, pelleted by centrifugation, and resuspended in 300 μl PBS.Virus was released from the cells by three cycles of freeze/ thaw asdescribed above. One hundred microliters of crude virus stock wastransferred to a 1.5 ml tube, an equal volume of melted 2% low meltingpoint agarose was added, and the virus/agarose mixture was transferredinto a pulsed field gel sample block. When the agar worms weresolidified they were removed from the sample block and cut into threeequal sections. All three sections were transferred to the same 1.5 mltube, and 250 μl of 0.5M EDTA. 1% Sarkosyl, 0.5 mg/ml Proteinase K wasadded. The worms were incubated in this solution at 37° C. for 24 hours.The worms were washed several times in 500 μl 0.5×TBE buffer. and onesection of each worm was transferred to a well of a 1% low melting pointagarose gel. After the worms were added the wells were sealed by addingadditional melted 1% low melting point agarose. This gel was thenelectorphoresed in a Bio-Rad pulsed field gel electrophoresis apparatusat 200 volts, 8 second pulse times, in 0.5×TBE for 16 hours. The gel wasstained in ethidium bromide, and portions of agarose containing vacciniagenomic DNA were excised from the gel and transferred to a 1.5 ml tube.Vaccinia DNA was purified from the agarose using β-Agarase (Gibco)following the recommendations of the manufacturer. Purified vaccinia DNAwas resuspended in 50 μl ddH₂O. One microliter of each DNA stock wasused as the template for a Polymerase Chain Reaction (PCR) usingvaccinia TK specific primers MM428 and MM430 (which flank the site ofinsertion) and Klentaq Polymerase (Clontech) following therecommendations of the manufacturer in a 20 μl final volume. Reactionconditions included an initial denaturation step at 95° C. for 5minutes, followed by 30 cycles of: 94° C. 30 seconds, 55° C. 30 seconds,68° C. 3 minutes. Two and a half microliters of each PCR reaction wasresolved on a 1% agarose gel, and stained with ethidium bromide.Amplified fragments of diverse sizes were observed. When corrected forflanking vector sequences amplified in PCR the inserts range in sizebetween 300 and 2500 bp.

The vaccinia virus cDNA library was further characterized in terms ofthe representation of clones homologous to the murine alpha tubulinsequence. Twenty separate pools with an average of either 300, 900 or2,700 viral pfu from the library were amplified by infecting a monolayerof 143B tk− cells in the presence of BrdU. DNA was extracted from eachinfected culture after three days and assayed for the presence of analpha tubulin sequence by PCR with tubulin specific primers. Poissonanalysis of the frequency of positive pools indicates a frequency of onealpha tubulin recombinant for every 2000 to 3000 viral pfu. This is notsignificantly different from the expected frequency of alpha tubulinsequences in this murine tumor cell line and suggests representativeexpression of this randomly selected sequence in the vaccinia cDNAlibrary.

Discussion

Tile above-described tri-molecular recombination strategy yields closeto 100% viral recombinants. This is a highly significant improvementover current methods for generating viral recombinants by transfectionof a plasmid transfer vector into vaccinia virus infected cells. Thislatter procedure yields viral recombinants at a frequency of the orderof only 0.1%. The high yield of viral recombinants in tri-molecularrecombination makes it possible, for the first time, to efficientlyconstruct genomic or cDNA libraries in a vaccinia virus derived vector.In the first series of experiments a titer of 6×10⁶ recombinant viruswas obtained following transfection with a mix of 20 micrograms of Not Iand Apa I digested vaccinia vector arms together with an equimolarconcentration of tumor cell cDNA. This technological advance creates thepossibility of new and efficient screening and selection strategies forisolation of specific genomic and cDNA clones.

The tri-molecular recombination method as herein disclosed may be usedwith other viruses such as mammalian viruses including vaccinia andherpes viruses. Typically, two viral arms which have no homology areproduced. The only way that the viral arms can be linked is by bridgingthrough homologous sequences that flank the insert in a transfer vectorsuch as a plasmid. When the two viral arms and the transfer vector arepresent in the same cell the only infectious virus produced isrecombinant for a DNA insert in the transfer vector.

Libraries constructed in vaccinia and other mammalian viruses by thetri-molecular recombination method of the present invention may havesimilar advantages to those described here for vaccinia virus and itsuse in identifying target antigens in the CTL screening system of theinvention. Similar advantages are expected for DNA libraries constructedin vaccinia or other mammalian viruses when carrying out more complexassays in eukaryotic cells. Such assays include but are not limited toscreening for DNA encoding receptors and ligands of eukaryotic cells.

EXAMPLE 2 Induction of Cytotoxic T Cells Specific for Human Tumors inHLA and Human CD8 Transgenic Mice

In this example, HLA and human CD8 transgenic mice were tolerized with anon-tumorigenic, immortalized normal human cell line that does notexpress costimulator activity for murine T cells and were subsequentlyimmunized with B7 (costimulator) transfected tumor cells derived by invitro mutagenesis or oncogene transformation from that same normal cellline. The HLA transgene permits selection of a high affinity,HLA-restricted T cell repertoire in the mouse thymus. In addition, ahuman CD8 transgene is required because murine CD8 does not interactefficiently with human class I MHC. Subsequent to immunization with B7transfected tumor cells, splenic CD8+ T cells are isolated andstimulated again in vitro in the absence of costimulation withnon-tumorigenic, immortalized human cells. Two pathways of toleranceinduction for antigens shared by the tumorigenic and non-tumorigeniccell lines may be activated through these manipulations. As known tothose skilled in the art, antigen exposure in very young mice favorstolerance induction by mechanisms that may include both clonal deletionand induction of T cell anergy. Further, restimulation of activated Tcells through their antigen-specific receptors in the absence ofcostimulator activity induces apoptotic elimination of those T cells.This immunization regimen enriched for tumor-specific CTL that did notcrossreact with the homologous normal cells.

A series of tumor cell lines were used that were all derived from asingle immortalized, non-tumorigenic cell line. The non-tumorigeniccells were used to induce tolerance to the large number of normal humanproteins that are also expressed in tumor cells. Availability of a panelof tumors independently derived from the same normal cells by diversecarcinogens or oncogene transformation makes it possible to filter outantigenic changes that are carcinogen specific or that may arise byrandom genetic drift during in vitro propagation of a tumor cell line.

Cytotoxic T cells specific for human bladder tumor cell lines wereinduced and isolated from (HLA-A2/K^(h)×human CD8)F₁ hybrid doubletransgenic mice that had been tolerized to the normal cell line fromwhich the tumors derive. Neonatal mice were injected intraperitoneallywith 5×10⁶ non-tumorigenic SV-HUC. Seven weeks later they were immunizedwith 5×10⁶ B7.1 transfected ppT11.B7 tumor cells. ppT11 is one ofseveral independent tumor cell lines derived from SV-HUC by in vitrocarcinogenesis (Christian, et al., Cancer Res. 47:6066-6073 (1987);Pratt, et al., Cancer Res. 52:688-695 (1992); Bookland, et al., CancerRes. 52:1606-1614 (1992)). One week after immunization, spleen wasremoved and a single cell suspension prepared. CD8 positive T cellprecursors were enriched on anti-Lyt-2 coated MACS (Magnetic cellsorting beads) as recommended by the manufacturer (Miltenyi Biotech,Sunnyvale, Calif.). 1.5×10⁶ CD8 enriched T cells were then restimulatedin vitro with 4×10⁵ SV-HUC in 3 ml of RPMI 1640+10% fetal bovine serum.The rationale is that any SV-HUC specific T cells that escape neonataltolerance induction and are activated in vivo by stimulation withcrossreactive determinants of ppT11.B7, might now be induced to undergoapoptosis by restimulation in vitro with costimulator activity negativeSV-HUC cells. After 24 hours, T cells are again stimulated with ppT11.B7in the presence of 2000 Units/ml of recombinant murine IL-6. On day 7the cycle of SV-HUC stimulation followed 24 hours later by restimulationwith ppT11.B7 is repeated. This second round of stimulation withppT11.B7 is carried out in the presence of 10 nanogram/ml recombinantmurine IL-7 and 50 Units/ml recombinant murine IL-2. CTL activity isdetermined 5 days later by standard chromium release assay from labeledtargets SV-HUC, ppT11.B7 and YAC-1.a cell line sensitive to non-specifickilling by murine NK cells. The results in Table 4 show that CTL fromppT11.B7 immunized mice that were not previously tolerized to SV-HUC areequally reactive with SV-HUC and ppT11 target cells. In contrast,following neonatal tolerization with SV-HUC, cytolytic T cells at aneffector:target ratio of 5:1 are significantly more reactive withppT11.B7 tumor cells than with SV-HUC. Note that B7 costimulator.activity is not required at the effector stage as similar results areobtained with B7 transfected or non-transfected target cells.

TABLE 4 Tumor-specific response in (HLA-A2/K^(b) × human CD8)F₁ hybridtransgenic mice neonatally tolerized with SV-HUC parental cells and thenimmunized with B7 costimulator transfected ppT11.B7 human bladder tumorcells. Tolerogen: None SV-HUC Immunogen: ppT11.B7 ppT11.B7Effector:Target ratio Target 5:1 10:1 2:1 5:1 SV-HUC 29 68 14 19ppT11.B7 14 70 17 51 YAC-1 6 6 nd 3 nd = not done

The significance of this experimental protocol is that it offers a meansof selecting murine, HLA-restricted cytolytic T cells specific for humanepithelial tumor cells. As noted previously, it has proved exceedinglydifficult to isolate such T cells directly from either patient PBL ortumor infiltrating lymphocytes of tumors other than melanoma and perhapsrenal cell carcinoma. In addition, as emphasized below, this samestrategy can be implemented in two stages. Differentially immunogenicmolecules of the human tumor can first be identified employingtumor-specific CTL restricted to a variety of different animal MHC.These antigens can, as described in Example 12, subsequently becharacterized in human subjects or transgenic mice for the ability to beprocessed and presented in association with different human HLA types.An advantage of this two stage approach is that numerous different MHCmolecules are available in a variety of inbred strains and these can beemployed to capture an equally broad range of tumor-specific immunogenicpeptides in the initial screening.

EXAMPLE 3 High-Throughput Strategy for Selection of DNA Recombinantsfrom a Library that Encodes the Target Epitopes of Specific Cytotoxic TCells

In this example, a model system was assayed to determine the level ofenrichment that can be obtained through a procedure that selects for DNArecombinants that encode the target epitopes of tumor specific cytotoxicT cells.

Methods and Results

A specific vaccinia recombinant that encodes a well characterizedovalbumin peptide (SIINFEKL) (SEQ ID NO:10) was diluted withnon-recombinant virus so that it constituted either 0.2%, 0.01%, or0.001% of viral pfu. This ovalbumin peptide is known to be processed andpresented to specific CTL in association with the murine class I MHCmolecule H-2K^(b). An adherent monolayer of MC57G cells that expressH-2K^(b) were infected with this viral mix at m.o.i.=1 (approximately5×10⁵ cell/well). MC57G cells do not themselves express ovalbuminpeptide. but do express H-2K^(b), which allows them to associate withand present ovalbumin peptide to the T cells.

Following 12 hours of infection with the recombinant vaccinia virusexpressing ovalbumin peptide, ovalbumin peptide-specific CTL, derived byrepeated in vitro stimulation of ovalbumin primed splenic T cells withthe immunodominant ovalbumin SIINFEKL peptide. were added for 30 min.

During this time, some of the adherent cells infected with a recombinantparticle that leads to expression of the ovalbumin peptide interactedwith a specific cytotoxic T cell and underwent a lytic event. Cells thatunderwent a lytic event were released from the monolayer. After 30 min,the monolayer was gently washed, and the floating cells and theremaining adherent cells were separately harvested.

Virus extracted from each cell population was titred for the frequencyof ovalbumin recombinant viral pfu. Virus extracted from floating cellswas then used as input to another enrichment cycle with fresh adherentMC57G cells and ovalbumin peptide-specific CTL. It was observed that,following enrichment of VVova to greater than 10% of total virus,further enrichment of the recombinant virus was accelerated if them.o.i. in succeeding cycles was reduced from 1 to 0.1. The results,presented in Table 5, demonstrate marked enrichment of VVova recombinantvirus from an initial concentration of 0.2% to 49% or from 0.01% to 39%in 5 enrichment cycles and from 0.001% to 18% in 6 enrichment cycles.Note that with 5×10⁵ adherent MC57G cells per well and m.o.i=1, aninitial concentration of 0.001% VVova recombinant virus is equivalent,on average, to seeding only 5 recombinant pfu among 5×10⁵ wild typevaccinia virus in a single culture well. A very substantial enrichmentis achieved even under these conditions.

TABLE 5 Multiple Cycles of Enrichment for Vvova % VVova in Floatingcells* Enrichment cycle # Exp. 1 Exp. 2 Exp. 3 moi = 1 0 0.2 0.01 0.0011 2.1 0.3 nd 2 4.7 1.1 nd 3 9.1 4.9 nd 4 14.3 17.9 1.4 5 24.6 3.3 6 18.6moi = 0.1 5 48.8 39.3 *% Vvova = (Titer with BrdU/Titer without BrdU) ×100 nd = not determinedDiscussion

The above-described selection method for isolating DNA clones thatencode target epitopes of specific cytotoxic T cells from a virallibrary is far more efficient than existing methods for accomplishingthis same goal. Prior to the present invention, the most widely employedmethod requires transfection of numerous small pools of recombinantplasmids into separate target populations in order to assay T cellstimulation by a minor component of some pool. Because this requiresscreening out many negative plasmid pools, it is a far more laborintensive procedure than the positive selection method described herein.For a given investment of resources, the method described here candetect positive DNA clones that occur at a much lower frequency thanwould otherwise be possible. The design principle of this strategy canbe directly extended to screening and selection of DNA clones withspecific antibodies as well as with CTL.

EXAMPLE 4 Identification of Potential Tumor-Specific Antigens that areDifferentially Expressed in Tumors

Identification of genes that are differentially expressed in humantumors, cancers, or infected cells could facilitate development ofbroadly effective human vaccines. Most methods for identification ofdifferential gene expression are variations of either subtractivehybridization or the more recently described differential displaytechnique.

Representational difference analysis (RDA) is a subtractivehybridization based method applied to “representations” of totalcellular DNA (Lisitsyn, N. and N., M. Wigler, “Cloning the differencesbetween two complex genomes,” Science 259:946-951 (1993)). Thedifferential display methods of Liang and Pardee (Science 257:967-971(1992)) employ an arbitrary 10 nucleotide primer and anchored oligo-dTto PCR amplify an arbitrary subset of fragments from a more complex setof DNA molecules. As described below (Example 4), we have modifieddifferential display to enhance the efficiency with which differentiallyexpressed genes can be identified. In this example we illustrate howapplication of these methods to a related set of tumors independentlyderived from a single non-tumorigenic, immortalized cell linefacilitates identification of tumor-specific gene products.

Experiments described by Sahasrabudhe, et al., (J. Immunology151:6302-6310 (1993)), focused on a set of murine tumor cell lines, allof which were independently derived from a single cloned,non-tumorigenic BALB/c embryonic fibroblast cell line. These tumors wereof particular interest because they are known to share animmunoprotective antigen. The goal of these experiments was to arrive ata molecular definition of that shared tumor antigen. The readyavailability of tumor cells, as well as the normal cells from which theywere derived, was exploited for efficient analysis of differential geneexpression and tumor immunogenicity by the methods described below.

The availability of multiple tumors independently derived from the samenormal cells by diverse carcinogens (or oncogene transformation) alsomakes it possible to filter out antigenic changes that are carcinogenspecific or that may arise as a result of random genetic drift during invitro propagation of tumor cells. (See Example 10, where a series ofhuman tumor cell lines is described that satisfy the requirements ofthis analysis).

The relationship between the process of transformation and expression ofshared tumor rejection antigens was investigated by characterizing theimmunological relationships among a series of murine tumors (BCA 34, BCA39, BCA 22, and BCB 13) independently derived from B/C-N7.1C.1, acontact inhibited, non-tumorigenic clone of a continuous fibroblast cellline derived from a BALB/c fetus (Collins, et al., Nature 299:167(1992); Lin, et al., JNCI 74:1025 (1985)). Although the proximal causeof tumor transformation may have been a carcinogen induced mutation,this model afforded the opportunity to determine if the process oftransformation is also associated with expression of a limited number ofshared antigens.

As reported by Sahasrabudhe, et al. (J. Immunology 151:6302-6310(1993)), immunological analysis demonstrates that three of four B/c.Nderived tumors confer crossprotective immunity against each other.Concordant with the in vivo cross-protection data, cytolytic T cellclones from mice immunized with one of the immunologically relatedtumors specifically lyse all three immunologically related tumors but,importantly, do not react with the parental B/c.N cells or with theimmunologically independent BCB 13 tumor. The observation ofimmunological cross-reactivity among a group of tumors independentlyderived from a cloned non-tumorigenic parental cell line stronglysuggests that a non-random transformation associated process gives riseto recurrent expression of the same tumor antigen(s). Two methods foranalyzing differential gene expression, representational differenceanalysis (RDA) and modified differential display, were employed toisolate cDNA that might encode the relevant tumor antigen(s).

Representational Difference Analysis (RDA)

The PCR SELECT™ variation of RDA is marketed by Clontech (Palo Alto,Calif.). The following general protocol outlined in the text and in FIG.3 is a: summary of the manufacturer's recommendations. cDNA issynthesized from both a tracer (represented by BCA 39 tumor mRNA) and adriver (represented by parental B/c.N mRNA). “Representations” of bothtracer and driver cDNA are created by digestion with RsaI which cuts thefour-base recognition sequence GTAC to yield blunt end fragments.Adaptors, which eventually serve as primer sites for PCR, are ligated tothe 5′ ends of only the tracer cDNA fragments (FIG. 3). Two aliquots oftracer representation are separately ligated with two differentadapters. A series of two hybridizations are carried out. In the firstset of hybridizations, each adapter ligated tracer sample is denaturedand hybridized with a ten fold excess of the denatured representation ofdriver cDNA for 8 hours. Under these conditions re-annealing of allmolecules is incomplete and some of both the high and low copy moleculesremain single stranded. Since re-annealing rates are faster for moreabundant species, this leads to normalization of the distributionthrough relative enrichment of low copy number single strandedmolecules. The two hybridization reactions with each of the differentadapter ligated tracer cDNA representations are then combined withoutfractionation or further denaturation but with addition of more freshlydenatured driver in a second hybridization reaction that is allowed toproceed further to completion, approximately 20 hours.

An aliquot of the products from the second hybridization is used as atemplate for a high stringency PCR reaction, using the known sequencesat the 5′ ends of the ligated adapters as primers. The key here is thatonly tumor tracer sequences that 1) remain single stranded through thefirst hybridization and 2) hybridize to a complementary tracer sequenceligated to the alternate adapter in the second hybridization can beexponentially amplified during PCR. This excludes both tracer and driverspecies that either remain single stranded or that have hybridized toexcess driver (since they have a complementary primer at only one orneither end of the molecule), as well as tracer sequences that hybridizeto a molecule with the same adapter (because the adapters are longerthan the primers and hybridize to their own complement with higheraffinity when it is present on the opposite end of a denatured singlestranded molecule—a reaction termed “Suppression PCR” by Clontech).Finally, a second high stringency PCR is performed using nested primersbuilt into the adapters so as to further reduce background and enrichfor differentially expressed sequences. The products of the second PCRare electrophoresed and visualized on an agarose gel. Individual bandsare excised and subcloned for further analysis.

Representational Difference Analysis of Genes that Encode PotentialTumor Immunogens

This example describes how the PCR SELECT™ cDNA subtraction method(Clontech Laboratories) was successfully employed to identify a strongcandidate for the shared tumor antigen in a set of immunologicallyrelated murine tumors.

As shown in FIG. 4, subtraction of a fragmented representation of normalcell cDNA from a similar representation of BCA 39 tumor cDNA resulted inidentification of a series of seven clearly distinguishable subtractionproducts ranging in size from approximately 300 to 2200 base pairs. Toconfirm that these DNA fragments were indeed differentially expressed,each band was cloned into Bluescript plasmid (Stratagene) and the DNAinserts of at least 5 colonies from each band were analyzed by Northernblot hybridization to RNA of the five different cell lines: the parentalcells, the three immunologically crossreactive and the onenon-crossreactive tumor cell line. Representative results for clone 3fderived from RDA band 1 are shown in FIG. 5A.

The probe hybridized to at least three transcripts in the BCA 22, 34 and39 tumor mRNA. Expression of these transcripts is unique to these threeimmunologically crossreactive tumors. Minimal hybridization is detectedwith RNA of the parental B/c.N cells or of the non-crossreactive BCB 13tumor. Similar results were obtained in four Northern blots withindependent RNA preparations. The integrity and relative loading of RNAsamples was determined by hybridization to a fragment of the mouse G3PDHgene (FIG. 5B).

The sequence of clone 3f was determined and found to be stronglyhomologous to a portion of the sequence of a murine intracisternal typeA particle (IAP element) (Aota. et al., Gene 56:1-12 (1987)). IAPs areendogenous retrovirus-like particles that localize to the cisternae ofthe endoplasmic reticulum. They are non-infectious because they do notencode functional packaging proteins; the potential env region of thesequence contains many conserved stop codons (Kuff and Lueders, Adv.Cancer Res. 51:183-276 (1988)). Most IAPs do encode a 73 kDa major gagprotein, and a pol polypeptide with some reverse transcriptaseproperties (Wilson and Kuff, Proc. Natl. Acad. Sci. USA 69:1531-1536(1972)). Expression of IAP transcripts has been described in variousmouse primary tumors (including plasmacytomas, papillomas, carcinomas,mammary tumors, sarcomas, hepatomas) and established mouse tumors andcell lines (including Friend erythroleukemias, myelomonocytic leukemia,T lymphomas, myelomas). Although expression in normal thymus may beelevated, only very low levels of expression are detected in most normalmouse somatic tissues (Kuff and Lueders, Adv. Cancer Res. 51:183-276(1988)).

Characterization of Differentially Expressed Gene Sequence from RDA

Semi-quantitative PCR is a more sensitive test for differentialexpression than Northern Blot analysis. Clone 3f sequence specificprimers were used to amplify full length oligo-dT primed cDNA from boththe BCA 39 tumor and the parental cell line. Amplification with mousetubulin primers was used to normalize the amount of template between thetwo cell lines. Equal aliquots of each template were amplified through avariable number of PCR cycles. In each case an estimate of the relativetemplate concentration was derived by fitting a line to the portion ofthe amplification curve in which product increases exponentially withcycle number. The assumption is that in this region yield is a linearfunction of (initial template concentration)*(a^(n)) where a=averageamplification per cycle in that PCR region, usually between 1.5 and 1.8,and n=cycle number. It was determined that expression of the 3f fragmentis at least 7 times greater in the BCA39 tumor cDNA relative to theparental B/c.N.

Differential expression in tumor RNA was confirmed for the inserts of 12additional clones derived from the six other RDA bands. Northernanalysis showed the identical hybridization pattern characteristic ofIAP transcripts as observed for clone 3f. The sequence of each clone wasdetermined and found to be homologous to other regions of the IAPgenome. A map of the relative position of 10 unique RDA clones is shownin FIG. 6. It can be seen that cumulatively these inserts cover most ofthe IAP genome.

It is particularly striking that expression of these IAP sequences isshared among the three immunologically crossreactive tumors (BCA 39, BCA34, and BCA 22) but is absent or very low in both the B/c.N parentalcells and the immunologically unrelated BCB 13 tumor. An IAP epitope is,therefore, a strong candidate for this shared tumor antigen. Experimentsare in progress to transfect the different RDA clones into antigennegative B/c.N cells which will then be tested for sensitization tolysis by tumor-specific CTL. Transcriptional activation of endogenousretroviral elements including IAP may represent a new class of sharedtumor rejection antigens. It has been reported (de Bergeyck, et al.,1994, Eur. J. Immunol. 24:2203-2212) that the tumor antigen LEC-A on themurine LEC spontaneous leukemia is also encoded by the gag gene of anIAP element. Recently, a tumor rejection antigen of a murine colontumor, CT26, was found to be encoded by another type of endogenousretrovirus, a type C particle (Huang, et al., Proc. Natl. Acad. Sci. USA93:9730-9735(1996)). Retroviral-like elements are also present in thehuman genome: expression of the pol gene has been detected in humanbreast (Moyret, et al., Anticancer Res. 8:1279-1283 (1988)) andcolorectal carcinomas (Moshier. et al., Biochem. Biophys. Res. Commun.139:1071-1077 (1986)), and antibody to the gag gene product has beenreported in the sera of patients with human seminoma (Sauter. et al., J.Virol. 69:414-421 (1995)) and renal cell carcinoma (Wahlstrom, et al.,Lab. Invest. 53:464-469 (1985)).

Modified Differential Display of Genes Encoding Potential TumorImmunogens

In the following example, the differential display methods of Liang andPardee (1992, Science 257:967-971) were modified to improve resolutionof DNA fragments and reduce the frequency of false positives.

The differential display method as originally described by Liang andPardee (Science 257:967-971 (1992)) employs an arbitrary 10 nucleotideprimer and anchored oligo-dT to PCR amplify an arbitrary subset offragments from a more complex set of DNA molecules. In principle,differences among the fragments generated from normal and tumor celllines should reflect differences in gene expression in the two celltypes. In practice, this method sometimes works well but often givesrise to numerous false positives. That is, bands which appear to bedifferentially displayed are, upon further characterization, found notto be differentially expressed. This is presumably due to variable PCRamplification of individual species in complex populations and arelatively high background that can obscure less prominent bands. Sinceconsiderable effort is required to establish differential expression,these endemic false positives are costly in terms of efficiency andproductivity. A single arbitrary primer may also be used fordifferential display, as described by Welch et al. (3,4). Use of singleprimers does, however, require synthesis of a much larger set ofindependent primers to achieve the same coverage of a complex cDNApopulation.

Hence, there exists a need for improved differential display methodsthat improve resolution of DNA fragments and that reduce the frequencyof false positives.

In order to improve the resolution of fragments and reduce the frequencyof false positives, a second arbitrary primer was substituted for theanchored oligo-dT employed in PCR amplification. This results in fewerDNA products in each PCR reaction so that individual DNA fragments canbe more reliably resolved on sequencing gels.

Because each subset of fragments generated in this modified differentialdisplay protocol is a smaller representation of total cDNA, more primerpairs are required for adequate sampling. Employing the negativebinomial distribution, it can be predicted that if 12 independentprimers are utilized in all 66 possible primer pair combinations thereis a greater than 85% probability that for an average size eukaryoticcDNA at least one primer pair will amplify a representative PCR fragmentof size ≧70 bp.

Table 6 lists the sequences of the 12 arbitrary decamers from whichprimer pairs are selected for modified differential display. Thespecific primers were chosen on the basis of their sequence diversity,3′ hybridization affinity, and minimal pair-wise hybridization.

TABLE 6 Arbitrary Primers For Modified Differential Display TAC AAC GAGG MR_1 (SEQ ID NO:11) GTC AGA GCA T MR_2 (SEQ ID NO:12) GGA CCA AGT CMR_5 (SEQ ID NO:13) TCA GAC TTC A MR_7 (SEQ ID NO:14) TAC CTA TGG C MR_8(SEQ ID NO:15) TGT CAC ATA C MR_15 (SEQ ID NO:16) TCG GTC ACA G MR_9(SEQ ID NO:17) ATC TGG TAG A MR_10 (SEQ ID NO:18) CTT ATC CAC G MR_11(SEQ ID NO:19) CAT GTC TCA A MR_12 (SEQ ID NO:20) GAT CAA GTC T MR_14(SEQ ID NO:21) CTG ATC CAT G Ldd1 (SEQ ID NO:22)

A separate cDNA synthesis reaction with 0.1 μg polyA-RNA and SuperscriptII Reverse Transcriptase (Gibco/BRL) is carried out with each primer.Five percent of the cDNA product made with each member of a primer pairis mixed together with that primer pair for amplification in 30 PCRcycles using Klen Taq Polymerase Mix (Clontech). The PCR primers areused for cDNA synthesis to avoid the 3′ bias imposed by oligo-dT primedcDNA synthesis. The relative orientation of the two primers in cDNA israndomized by carrying out a separate synthesis with each primer. ThesecDNA can be mixed in the same combinations as the primers chosen for PCRamplification. PCR amplified cDNA fragments are resolved on 6%acrylamide gels and dried for autoradiography. Those bands which aredifferentially displayed in at least 2 tumor samples and not in theparental cells are cut out and rehydrated. An aliquot (1/5) of the DNArecovered is reamplified using the same primer set and the same PCRconditions but without addition of isotope. This second PCR product isresolved on 1% agarose and individual bands are recovered by incubationwith β agarase I (Gibco/BRL). Each DNA fragment recovered is cloned byblunt end ligation into the pcDNA3.1/Zeo (+) phagemid vector(Invitrogen). Since it is possible that a single band may include morethan one molecular species, at least 4 different transformants with aninsert of appropriate size are picked for further characterization.Northern analysis, RNase protection assays and semi-quantitative PCR areemployed to confirm differential expression.

In murine tumor cell lines, it was observed that many moredifferentially expressed gene fragments appear to be identified bydifferential display than by RDA. In addition, RDA fragments givepositive results on Northern blots exposed for only a few hours. Incontrast, fragments identified by differential display often do not givea signal on Northern blots even after several days. Differentialexpression was, in this case, confirmed by Rnase protection andsemi-quantitative PCR with sequence specific primers. These observationsare consistent with the theoretical expectation that, because of thedifficulty of driving hybridization of low abundance cDNA to completion,such sequences will be more readily identified by PCR based differentialdisplay than by hybridization based RDA. There may, in addition, beanother reason for the greater sensitivity of modified differentialdisplay. It has been reported (Meyuhas and Perry, Cell 16:139-148(1979)) that mRNA species of low abundance are on average twice the sizeof smaller, more stable and more abundant mRNA species. It is,therefore, more likely that both members of a pair of arbitrary primerswill hybridize to and detect differentially expressed cDNA from thelonger (average 4.9 kb) very diverse 80% of mRNA species that arerepresented by very few copies per cell than from the shorter (average 2kb) 20% of mRNA species that are more abundantly expressed.

In preliminary experiments, an average of three differentially displayedbands were identified for each pair of primers. With a total of 66primer pairs generated from all possible combinations of 12 independentprimers, approximately 200 gene fragments could be identified. In somecases multiple fragments may derive from the same gene. FIG. 7 shows thepattern of differential display fragments observed with one pair ofarbitrary decamers, MR_(—)1 (TAC AAC GAG G) (SEQ ID NO: 11) and MR_(—)5(GGA CCA AGT C) (SEQ ID NO: 13). A number of bands can be identifiedthat are associated with all four tumors but not with the parentalcells. This distribution is unrelated to the immunogenicity of the tumorcells, since only three of the four tumors are immunologicallycrossreactive. In contrast to the differentially expressed bandsidentified by RDA, which gave positive results on the Northern blotsexposed for only a few hours, fragments identified by differentialdisplay did not give a signal on Northern blots even after several days.Differential expression of the differential display fragments can,however, be confirmed by RNase protection assays or by semi-quantitativePCR with sequence specific primers. An example is shown in FIGS. 8A and8B the results of an RNase protection assay with clone 90 fromdifferential display band 9. This sequence, which has no significanthomology to entries in the GenBank database, is expressed in all fourtumor lines but not in the parental B/c.N.

As discussed above, we attribute this striking difference in the resultsof RDA and differential display to the greater sensitivity of the PCRbased modified differential display as compared to the hybridizationbased RDA method. Based on the pattern of expression in the differenttumor and normal cell lines, it appears that the shared tumor antigendetected following direct immunization of mice with syngeneic tumorcells may be encoded by a more abundantly expressed IAP gene. Themethods described in this example can be used to determine whether theproducts of the less abundantly expressed genes identified by modifieddifferential display represent potential cryptic tumor antigens.

Selection of Full Length cDNA Encoding Potential Tumor Immunogens

This section presents methods for facilitating selection ofcorresponding full length cDNAs from fragments of differentiallyexpressed genes identified by representational difference analysis or bymodified differential display (FIG. 9). A single stranded biotinylatedprobe is synthesized from isolated cDNA fragments and is used to selectthe longer cDNA that contain a complementary sequence by solutionhybridization to single stranded circles rescued from a phagemid tumorcDNA library. This method is especially well-suited to the use of DNAfragments isolated by the modified differential display method employingtwo arbitrary primers. The same arbitrary primers employed for PCRamplification of a given fragment in differential display can bemodified to generate a single stranded hybridization probe from thatfragment. This avoids the need to sequence, select and synthesize a newpair of fragments specific primers for each new fragment of interest.

i) The two oligonucleotides of a pair of PCR primers employed indifferential display are modified: (biotin-dT)-dT-(biotin-dT) isincorporated at the 5′ end of one primer and a phosphate is incorporatedat the 5′ end of the second primer. These modified primers areincorporated by PCR into the two strands of a differential displayfragment that was selected following the original PCR amplification withthe same unmodified arbitrary primers. From this double stranded PCRproduct, the strand labelled with a 5′ phosphate is digested with λexonuclease to generate a single stranded biotin-labeled probe.

ii) Single stranded (ss) DNA circles are rescued from a phagemid cDNAlibrary using the M13K07 packaging defective phage as helper virus. Thislibrary is constructed in the pcDNA3.1/Zeo(+) phagemid (Invitrogen,Carlsbad, Calif.) with insertion of (ApaI)oligo-dT primed cDNA betweenthe Apa I and Eco RV restriction sites. A key manipulation to achievethe efficient ligation necessary for construction of a high titer cDNAlibrary is to insure that cDNA inserts are 5′ phosphorylated by treatingwith T4 polynucleotide kinase prior to ligation. The biotin-labeledsingle stranded probe generated from the differential display fragmentis hybridized in solution to the ssDNA circles of the phagemid library.The biotin-labeled hybridization complexes can then be separated fromunrelated ssDNA on streptavidin magnetic beads and the ss circles elutedfor further analysis (FIG. 9).

As a test of this enrichment method, a model plasmid mix was preparedthat included 1% of a specific arbitrarily selected recombinant clone,3f IAP. A biotinylated ss-probe was prepared from the 3f RDA fragmentand used to select single stranded phagemid circles from the 1% plasmidmix. Following elution from streptavidin beads, the single strandedcircles were hybridized to a sequence specific oligonucleotide in orderto prime synthesis of the second plasmid strand prior to bacterialtransformation. Plasmid DNA was prepared from 63 transformed colonies.63 of 63 of these plasmid preparations expressed the target 3F IAPinsert. This method therefore appears to be very efficient.

The same method appears to work with similar efficiency in the morestringent case of a differential display fragment (B4) representing apreviously unidentified sequence that is expressed in all four murinetumors at a concentration approximately 10 fold greater than in thenon-tumorigenic parental cells. 5 out of 5 transformants randomly pickedfollowing selection of single strand circles with the 200 bp B4 DNAfragment had longer inserts that were positive by PCR with sequencespecific primers. This method therefore appears to be very efficient.

EXAMPLE 5 Independent Human Tumor Cell Lines Derived from aNon-Tumorigenic, Immortalized Cell Line

The following example describes a set of human tumors independentlyderived by different carcinogens or oncogene transformation from thesame cloned, non-tumorigenic parental cell line. As in the previousexamples of the use of RDA and modified differential display foridentification of gene products differentially expressed in murinetumors, the availability of related normal and tumor cell lines hasconsiderable advantages for the molecular and immunological analysis ofpotential cancer vaccines. This not only provides a readily availablesource-of normal control cells and RNA, but also makes it possible tofocus on molecular features that are carcinogen independent and, sincethey are shared by multiple independent tumors, are unlikely to be theproducts of random genetic drift during in vitro propagation.

A set of human uroepithelial tumors have been derived in the laboratoryof Dr. Catherine Reznikoff (University of Wisconsin, Madison) from anSV40 immortalized human uroepithelial cell line, SV-HUC, that is itselfcontact inhibited, anchorage dependent and non-tumorigenic in nude mice(Christian, et al., Cancer Res. 47: 6066-6073 (1987)). A series ofindependent tumor cell lines were derived by either ras transformation(Pratt, et al., Cancer Res. 52:688-695 (1992)) or in vitro mutagenesisof SV-HUC with different carcinogens including some that arebladder-specific (Bookland, et al., Cancer Res. 52:1606-1614 (1992)).Transformed cells were initially selected on the basis of altered invitro growth requirements and each was shown to be tumorigenic in nudemice. A subset of these tumors is selected that retain the phenotype oftransitional cell carcinoma. Table 7 lists the parental cells and thecarcinogens employed to derive these 5 tumor lines in vitro. Asystematic program is undertaken to 1) identify full length cDNAdifferentially expressed in these tumors and 2) to test theimmunogenicity in HLA and human CD8 transgenic mice of these cDNAproducts cloned into a vaccinia virus expression vector.

TABLE 7 Human Uroepithelial Cell Lines Acquired from Dr. Catherine A.Reznikoff, University of Wisconsin Clinical Caricer Center Parental LineImmortalization SV-HUC SV40 immortalized normal bladder epithelial cellsTumor Line Carcinogen or Oncogene transformation MC pT73-methylcholanthrene MC ppT11-A3 3-methylcholanthrene followed by4-aminobiphenyl MC ppT11-HA2 3-methylcholanthrene followed byN-hydroxy-4-acetylaminobiphenyl HA-T2 N-hydroxy-4-aminobiphenylSV-HUC/ras-T EJ/ras

Experiments apply both representational difference analysis and modifieddifferential display to identify gene fragments differentially expressedin the MC ppT11-A3 tumor (ppT11A3) relative to the parental SV-HUC. Alldifferentially expressed fragments are tested by Northern analysis andRNase protection assay for parallel expression in mRNA of the othertumor cell lines. Only those DNA clones expressed in at least 3 of the 5SV-HUC derived tumor cell lines are selected for furthercharacterization.

Similar analysis of tumor-specific gene products can be carried out withtumors derived from SV40 large T or HPV E6 or E7 immortalized cell linesrepresentative of other human tissues. Published examples include:prostatic epithelium (Parda et al., The Prostate 23:91-98 (1993)),mammary epithelium (Band et al., Cancer Res. 50:7351-73-57 (1990)), andbronchial epithelium (Gerwin et al., Proc. Natl. Acad. Sci. USA89:2759-2763 (1992); Klein-Szanto et al., Proc. Natl. Acad. Sci. USA89:6693-6697 (1992)).

EXAMPLE 6 Gene Expression in Fresh Patient Bladder Tumors

The above-described methods for identification of differentiallyexpressed genes require that both tumor and normal control cell mRNA bereadily available. The preceding section focuses on tumors derived invitro from immortalized cell lines, from which mRNA may be readilyobtained in large quantities.

In spite of the advantages of working with in vitro-derived tumors fromwhich mRNA may be readily obtained, it is necessary to address thepossibility that some transformation-associated gene expression might bemissed or, conversely, that some differential gene expression detectedmight not be transformation related. Although the normal control iscontact inhibited, anchorage dependent and non-tumorigenic, it is likelythat it has undergone some pre-neoplastic event that is the basis forcontinuous growth in vitro. Perhaps a greater concern is that extraneousgene expression associated with in vitro proliferation might beidentified. Two strategies to exclude such events are employed. First,genes are analyzed that are expressed in at least 3 of the 5 bladdertumor lines but that are not expressed in the in vitro adapted parentalcells. This will a) filter out any systematic gene expression selectedby in vitro growth, since this should be shared by the normal parentalcells; and b) identify any alterations in gene expression that arecarcinogen specific or that may arise as a result of random geneticdrift during in vitro propagation, since it is not expected that thesewould be shared by multiple independent tumors derived by diversecarcinogens (or oncogene transformation). Second, and most important,only those differentially expressed genes that can also be shown to beexpressed in multiple samples of fresh patient tumor material areselected for further characterization.

Patient tumor material together with normal bladder epithelium iscryopreserved following surgery. In comparison to some other carcinomas,normal tissue control is readily available from bladder cancer patients.Total RNA is extracted from frozen samples by the acid guanidiniumisothiocyanate method (Lee and Costlow, Methods in Enzymology152:633-648 (1987)). Following Dnase I treatment, polyA mRNA isfractionated on oligo dT beads and gene expression is analyzed byNorthern blot, RNase protection assay, and semi-quantitative RT/PCR. Foreach differentially expressed gene fragment identified in the in vitrotumor lines, expression of the gene is characterized in a panel of 20patient tumors and normal tissue controls. This sample size permits theestimation of the proportion of patients expressing the gene with astandard error no greater than 0.11% (SE=sqrt[p*(1−p)/n] where p=trueproportion and n=sample size. SE is maximal for p=0.5. at thatproportion, 10/20 patients, SE=±0.11; for any other value of p, SE issmaller.) Expression of some of these genes may be correlated in thedifferent tumor samples. This is useful because it creates thepossibility of multiple T cell epitopes that could associate withdifferent human MHC molecules.

The expression pattern is also determined, in other normal adult andfetal tissues, of any gene that is differentially expressed in bladdertumors relative to normal bladder epithelium. Total RNA or first strandcDNA prepared from over 30 different human normal adult or fetal tissues(Discovery Line™ RNA and Gene Pool™ cDNA, Invitrogen, Carlsbad, Calif.)is used. Expression in fetal but not normal adult tissue is particularlyinteresting and does not preclude consideration as an immunotherapeuticreagent. Expression of intermediate abundance species are determined byNorthern analysis. Low abundance species are quantitated by RNaseprotection assay and semi-quantitative PCR. Those sequences that arerecurrently expressed in tumors derived from multiple patients and whichhave the lowest relative expression in normal tissue are selected forfurther characterization as potential tumor-specific antigens.

EXAMPLE 7 The Use of Differentially Expressed Gene Products to GenerateCTLs Crossreactive with Authentic Tumors

To identify differentially expressed gene products that might becandidates for tumor immunotherapy, it is necessary to have a means ofdelivering the product for immunization in an environment in which Tcell responses to peptides associated with human HLA can be induced. Tcells induced by immunogenic products could then be tested forcrossreactivity on HLA compatible tumors that express the correspondingmRNA. This example describes the use of HLA and human CD8 transgenicmice for induction of T cell responses to peptides associated with humanHLA. If all these conditions are met: 1) the gene is differentiallyexpressed in multiple human tumors but not normal tissue counterparts;2) gene products are immunogenic in association with HLA; and 3) thespecific T cells induced are crossreactive on human tumor cells, thenthis would constitute key preliminary data preparative to initiation ofclinical vaccine trials.

To determine whether the products of differentially expressed genes areimmunogenic, groups of three (HLA-A2.1×huCD8)F₁ transgenic mice areimmunized intravenously with 5×10⁶ pfu of each specific recombinantvaccinia virus (Bennink and Yewdell, Current Topics in Microbiol. andImmunol. 163:153-178 (1990)). After at least two weeks, mice aresacrificed and CD8+ splenic T cells are enriched on anti-CD8 coatedmagnetic beads. CD8+ cytolytic precursors are restimulated in vitro withparental SV-HUC cells that are transfected with the recombinantdifferentially expressed gene previously isolated in the pcDNA3.1/Zeo(+)plasmid expression vector (Example 4). Substitution of the plasmidrecombinant in place of the vaccinia vector for restimulation in vitrois necessary to avoid a large vaccinia vector specific response. Afterfive days in vitro culture, cytolytic activity is determined by ⁵¹Crrelease from SV-HUC target cells transfected with either the specificrecombinant plasmid or a control ovalbumin gene recombinant.

This same cytolytic assay can be readily applied to determine whetherthe relevant CTL epitope is also presented by HLA compatible tumor cellsthat express the corresponding mRNA. If T cells are induced in(HLA-A2.1×huCD8)F₁ transgenic mice, HLA compatible targets include tumorcells that either express native HLA-A2.1 or that have been transfectedwith HLA-A2.1. The immunogenicity of differentially expressed geneproducts is established and it is determined whether there is acrossreaction with human tumor cells. This finding, together with thedemonstration that the same mRNA is expressed in multiple samples offresh patient tumors but not normal tissues (Example 6), is requiredprior to initiation of a clinical vaccine trial.

An important consideration for vaccine development is the extensivepolymorphism of humans class I HLA. As discussed above, an appealingstrategy is to target four major HLA subtypes, A2, A3, B7 and B44, thatprovide broad coverage across ethnic populations. Many peptides bind tomultiple members of a single subtype. If several CTL epitopes areidentified for each subtype, then this can greatly facilitateformulation of a broadly effective vaccine.

EXAMPLE 8 Introduction of Protective Immunity

It is desirable, especially in the case of cryptic tumor antigensencoded by low abundance mRNA, to determine whether a T cell response todifferentially expressed gene products confers protective tumorimmunity. Since a number of differentially expressed genes have beenidentified in the murine tumor model described above, such experimentsare carried out in mice.

It has previously been reported for this murine tumor model(Sahasrabudhe, et al., J. Immunology 151: 6302-6310 (1993)) that threeof four independently derived tumors are immunologically crossreactive.Many of the differentially displayed bands identified in these tumorsare, in contrast, present in all four tumors. It is, therefore, unlikelythat the genes from which these fragments derive are immunologicallydominant in animals inoculated with these tumors.

If it is shown that direct immunization with a recombinantdifferentially expressed gene does, nevertheless, confer protectiveimmunity, then this provides compelling evidence for the efficacy ofvaccination with a cryptic tumor antigen.

Groups of 5 mice of the BALB/c strain syngeneic to the murine tumors areimmunized with each vaccinia virus recombinant for a full length cDNAdifferentially expressed in all four murine tumor lines but not theparental B/c.N cells (FIG. 7). Each group of mice is assayed forinduction of protective immunity by challenge with a tumorigenicinoculum of 1×10⁶ BCA 39 tumor cells (Sahasrabudhe, et al., J.Immunology 151:6302-6310 (1993)). To determine whether protectiveimmunity correlates with relative quantitative expression, independentgene products are tested that represent different levels of differentialexpression as determined by semi-quantitative PCR.

EXAMPLE 9 Construction and Characterization of Vaccinia ExpressionVectors for Use in Vaccines

This example describes the construction and characterization of a newset of direct ligation vectors designed to be universally applicable forthe generation of chimeric vaccinia genomes. The aim was to modify thegenome of vNotI/tk so as to acquire direct ligation vectors which aremore universally useful. First, the insertion site was changed byplacing the sites for two unique restriction enzymes at the beginning ofthe thymidine kinase gene. This allows one to fix the orientation of theinsert DNA and eliminates the production of contaminating wild typegenomes after religation of viral arms. Second, in order to generate adirect ligation vector which would express high levels of protein, thethymidine kinase gene was preceded by a strong constitutive vacciniavirus promoter.

These new ligation vectors contain a pair of unique restriction sites,NotI and ApaI, to eliminate religation of poxvirus arms and fix theorientation of the insert DNA behind strongly expressing constitutivevaccinia promoters. The insertion cassette has been placed at thebeginning of the thymidine kinase gene in vaccinia to utilize-drugselection in the isolation of recombinants.

Materials and Methods

Plasmid Construction

Pairs of oligonucleotides were constructed which, when annealed,contained the 7.5k gene promoter(MM436:GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCCATGGGCCC (SEQ IDNO.:23) and MM437: GGCCGGGCCCATGGCGGCCGCGTGCAATAAATAGATCTAGTTTTTCAATTTTT(SEQ ID NO.:24)), or the synthetic EL promoter (MM438:GGCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAAGCGGCCGCCAT GGGCCC (SEQ IDNO.:25) and MM439:GGCCGGGCCCATGGCGGCCGCTTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTT (SEQ IDNO.:26)) and restriction sites for NotI and ApaI. The double-strandedoligonucleotides were annealed by ramping from 94° C. to 20° C. over twohours and ligated into the NotI site present in pJNotI/tk, a plasmidcontaining the HhindIII J fragment from vNotI/tk, resulting in plasmidsp7.5/tk and pEL/tk.

A Polymerase Chain Reaction (PCR) was performed on pBI221, a plasmidcontaining the E. coli gusA gene encoding for β-glucuronidase (β-glu),using primers MM440 (GGGAAAGGGGCGGCCGCCATGTTACGTCCTGTAGAAACC) (SEQ IDNO.27) and MM441 (GGGAAAGGGGGGCCCTCATfGTTTGCCTCCCTGCTG)(SEQ ID NO.:28),or MM440 and MM442 (GGGAAAGGGGCGGCCGCCTCATTGTTTGCCTCCCTGCTG) (SEQ IDNO.:29), and the resulting fragment was cloned into pCRII (TA cloningkit, Invitrogen). The plasmids were excised with NotI (MM440/MM442product) and cloned into pJNot/tk digested with NotI yieldingpJNot/tk-GUS, or excised with NotI and ApaI (MM440/MM441 product), andinserted into pEL/tk and p7.5/tk previously digested with ApaI and NotIyielding p7.5/tk-GUS and pEL/tk-GUS.

Pairs of oligonucleotides were constructed which, when annealed,contained the 7.5k gene promoter and the nucleotide sequence encodingfor a cytotoxic T-cell epitope for ovalbumin (11) (SIINFEKL; SEQ ID NO:10) (75ova:GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACCATGAGTATAATCAACTTTGAAAAACTGTAGTGA(SEQ ID NO.:30) and 75ovarv:GGCCTCACTACAGTTTTTCAAAGTTGATTAATACTCATGGTGCAATAAATAGATCTAGTT TTTCAATTTTT(SEQ ID NO.:31)) or the EL promoter and the peptide SIINFEKL (SEQ IDNO:10) (ELova:GGCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAACCATGAGTATAATCAACTTTGAAAAACTGTAGTGA (SEQ ID NO.:32) and ELovarv:GGCCTCACTACAGTTTTTCAAAGTTGATTATACTCATGGTTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTT (SEQ ID NO.:33)). The double-stranded oligonucleotideswere annealed by ramping from 94° C. to 20° C. over two hours andligated into the NotI site present in pJNotI/tk, a plasmid containingthe HindIII J fragment from vNotI/tk resulting in plasmids p7.5/tk-ovaand pEL/tk-ova.

Generation of Recombinant Viruses

Cells and viruses were maintained and manipulated as described by Earl,et al. (in Current Protocols in Molecular Biology, Ausubel, et al.,eds., Greene Publishing Associates/Wiley Interscience, New York (1991)).Recombinant viruses were made using homologous recombination byinfecting CV-1 cells at a multiplicity of infection (moi) of 0.05 andtwo hours later transfecting DNA into the infected cells usinglipofectamine (Life Technologies Incorporated) as suggested by tilemanufacturer. After 72 hours the cells were harvested and isolatedplaques were selected by passage in Hutk⁻ cells in the presence ofbromodeoxyuridine (Earl, et al., in Current Protocols in MolecularBiology, Ausubel, et al., eds., Greene Publishing Associates/WileyInterscience, New York (1991)) or HAT supplemented media (Weir, et al.,1982, Proc. Nat. Acad. Sci. USA, 79:1210-1214).

Vaccinia virus was generated from viral DNA by rescue with fowlpox virus(Scheiflinger, et al, Proc. Natl. Acad. Sci. USA 89:9977-9981 (1992)).Vaccinia virus was isolated from infected HeLa cells by banding andsedimentation in sucrose (Earl, et al., in Current Protocols inMolecular Biology, Ausubel, et al., eds., Greene PublishingAssociates/Wiley Interscience, New York (1991)). The purified virionswere treated with Proteinase K (Boehringer Mannheim) and gentlyextracted with buffer saturated phenol, phenol:chloroform (50:50), andchloroform before precipitation with 2.5 volumes of ethanol in 0.3Msodium acetate and resuspended in TE (10 mM TrisHCl, pH8.0. 1 mM EDTA(Earl, et al., in Current Protocols in Molecular Biology, Ausubel, etal., eds., Greene Publishing Associates/Wiley Interscience. New York(1991)). Confluent wells of BSC-1 cells from a 12 well dish wereinfected with fowlpox virus and after a two hour incubation at 37° C.were transfected with 0.6 μg full length vaccinia DNA usingLipofectamine (Life Technologies Incorporated) as suggested by themanufacturer. After 24, 48, and 72 hours the cells were harvested, lysedby three freeze-thaw cycles and screened by plaque assay on BSC-1 cells(Earl, et al., in Current Protocols in Molecular Biology, Ausubel, etal., eds., Greene Publishing Associates/Wiley Interscience, New York(1991)).

Generation of Recombinants Viruses by Direct Ligation

The 1.1 kB Eco RI/Eco RV restriction endonuclease fragment containingovalbumin from phbeta-Ova-neo (Pulaski, et al., 1996, Proc. Natl. Acad.Sci. USA, 93:3669-3674) was inserted into the EcoRI and EcoRV sites ofpBluescript KS+ (Stratagene)., generating pBS.ova. The DNA product froma Polymerase Chain Reaction (PCR) on pBS.ova using primers VV0LZ5(GCAGGTGCGGCCGCCGTGGATCCCCCGGGCTGCAGG) (SEQ ID NO.:34) and VVTLZ3(GTACCGGGCCCACAAAAACAAAATTAGTTAGTTAGGCCCCCCCTCGA) (SEQ ID NO.:35) wasdigested with ApaI and NotI (Life Technologies, Inc.), gel purified fromlow melting point agarose (Bio-Rad) using beta Agarase (LifeTechnologies, Inc.) following the recommendations of the manufacturer,and cloned into pBluescript KS+ that had been digested with NotI andApaI, generating pBS.VVova. A DNA fragment encoding ovalbumin wasexcised from pBS.VVova by digestion of this plasmid with ApaI and NotIand purified after electrophoresis through a low melting point agarosegel using beta Agarase. One microgram of purified vEL/tk DNA wasdigested with ApaI and NotI and centrifuged through a Centricon 100concentrator (Amicon) to remove the small intervening fragment. ThevEL/tk DNA arms and the DNA fragment encoding ovalbumin were ligatedovernight at room temperature, at a 4:1 (insert:virus) molar ratio, in30 microliters with 5 units T4 DNA Ligase. The ligation product wastransfected using lipofectamine (Life Technologies, Inc.) into a well ofconfluent BSC-1 cells from a 12 well plate two hours after infectionwith fowlpox virus at 1 pfu/cell. Three days later the cells wereharvested and isolated plaques were selected by passage in Hutk− cellsin the presence of bromodeoxyuridine (Earl, et al., in Current Protocolsin Molecular Biology, Ausubel, et al., eds., Greene PublishingAssociates/Wiley Interscience, New York (1991)).

Analysis of Viral DNA Genomes

BSC-1 cells were infected at high multiplicity of infection(moi)byvaccinia WR, vEL/tk, v7.5/tk, or vNotI/tk. After 24 hours the cells wereharvested and resuspended in Cell Suspension Buffer (Bio-Rad Genomic DNAPlug Kit) at 1×10⁷ cells/mil. An equal volume of2% CleanCut agarose(Bio-Rad) preincubated at 50° C. was added and the cell suspension wasformed into 100 μl plugs. After hardening at 4° C. the plugs weretreated as previously described to digest protein (Merchlinsky, et al.,J. Virol. 63:1595-1603 (1989)). The plugs were equilibrated in theappropriate restriction enzyme buffer and 1 mM PMSF for 16 hours at roomtemperature, incubated with restriction enzyme buffer, 100 ng/ml BovineSerum Albumin and 50 units NotI or ApaI for two hours at 37° C. (NotI)or room temperature (ApaI) prior to electrophoresis.

One well of a 6 well dish of BSC-1 was infected with v7.5/tk or vEL/tkat high multiplicity of infection (moi) and after 48 hours the cellswere harvested, pelleted by low speed centrifugation, rinsed withPhosphate-Buffered Saline (PBS), and the DNA was isolated using DNAzol(Gibco). The final DNA product was resuspended in 50 microliters of TE(10 mM TrisHCl, pH 8.0, 1 mM EDTA) and 2.5 microliters were digestedwith HindIII, HindIII and ApaI, or HindIII and NotI, electrophoresedthrough a 1.0% agarose gel, and transferred to Nytran (Schleicher andSchuell) using a Turboblotter (Schleicher and Schuell). The samples wereprobed with p7.5/tk (FIG. 11A) or pEL/tk (FIG. 11B) labeled with ³²Pusing Random Primer DNA Labeling Kit (Bio-Rad) in QuickHyb (Stratagene)and visualized on Kodak XAR film.

One well of a 6 well dish of BSC-1 cells was infected with v7.5/tk,vEL/tk, vNotI/tk, vpNotI, vNotI/lacZ/tk, or wild type vaccinia WR athigh multiplicity of infection (moi) and after 48 hours the cells wereharvested, pelleted by low speed centrifugation, rinsed withPhosphate-Buffered Saline (PBS), and the DNA was isolated using DNAzoI(Gibco). The final DNA product was resuspended in 50 microliters of TE(10 mM TrisHCl pH8.0. 1 mM EDTA) and used in a PCR (30 cycles, 1 minute94° C., 2 minutes 55° C. 3 minutes 72° C., MJ Research PTC-100) withprimers MM407 (GGTCCCTATTGTTACAGATGGAAGGGT) (SEQ ID NO.:36) and MM408(CCTTCGTTTGCCATACGCTCACAG) (SEQ ID NO.:37). The nucleotide sequence wasdetermined by ³⁵S sequencing using Sequenase Version 2.0 DNA SequencingKit (Amersham), and visualized after electrophoresis through 8%denaturing polyacrylamide gels by exposure to Bio-Max film (Kodak).

Determination of β-glucuronidase Activity

A well of BSC-1 cells from a 12 well plate was infected at an moi of 1with vNotI/tk-GUS, v7.5/tk-GUS and vEL/tk-GUS, the cells were harvested20 hours post infection, resuspended in 0.5 ml PBS, and disrupted bythree cycles of freeze-thawing. The extract was clarified by a shortmicrofuge spin (one minute, 14,000 rpm) and the supernatant was analyzedfor β-glu units as described by Miller, Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1972), as adapted for 96-well plates. The A₄₀₅ values were determinedoil a microplate reader (Dynatech MR3000) and the β-glu activity wasdetermined by comparison to β-glu (Clontech) standards analyzed in thesame assay.

Analysis of Cytoxic T Cell Response

Confluent monolayers of MC57G cells in wells of a 6 well plate wereinfected at an moi of 1 with vEL/tk. v7.5/tk-ova, vEL/tk-ova,vEL/tk-ovaFL clone 1, and vEL/tk-ovaFL clone 2 (vEL/tk-ovaFL are virusclones of full length ovalbumin generated by direct ligation). At 16hours post infection cells were harvested, labeled with 100 microcuries⁵¹Chromium (Dupont) for 1 hour at 37° C., and 10⁴ cells were added towells of a 96 well round bottom plate in quadruplicate. A sample ofuninfected MC57G cells incubated with 1 micromolar purified ova 257-264peptide was also incubated with ⁵¹Cr as a positive control and untreatedMC57G cells were used as a negative control. T cells specific for ova257-264 were added to target cells at ratios of 2:1 and 10:1. Cells wereincubated at 37° C. for 4 hours. supernatants were harvested, and ⁵¹Crrelease determined. Spontaneous release was derived by incubating targetcells with media alone and maximal release was determined by incubatingtarget cells with 5% Triton X 100. Percentage of specific lysis wascalculated using the formula: % specific lysis=((experimentalrelease-spontaneous release)/(maximal release-spontaneous release))×100.In each case the mean of quadruplicate wells was used in the aboveformula.

Results

Construction of Direct Ligation Vectors

The vaccinia WR genome is approximately 190 kilobases in length and richin A and T residues. The complete sequence of the vaccinia WR genome wasprovided by P. Earl of the Bernard Moss laboratory (Laboratory of ViralDiseases, NIAID, NIH, Bethesda, Md.). A restriction enzyme search of thecomplete sequence of the vaccinia WR genome using MacVector (IBI)revealed a lack of restriction sites for ApaI, AscI, BspI201, FseI,RsrII, SfiI, SrfI and SgfI. The ready availability of highly active andpure preparations of the enzyme as well as the generation of a staggeredend upon digestion led us to choose to use ApaI as the second site inconjunction with the NotI site already present in vNot/tk.

Vaccinia virus based expression vectors are most useful when the foreignprotein is expressed constitutively. The expression of foreign proteinsduring the early stage of viral replication is essential for cytotoxic Tcell response (Bennick, et al. Topics Microbiol. Immunol. 163:153-184(1990)) and high levels of total protein expression have been observedusing promoters active during the late stage of viral replication. Wedecided to incorporate the promoters corresponding to the constitutivelyexpressed 7.5k gene (Mackett, et al., J. Virology, 49:857-864 (1984))and a constitutively expressed synthetic promoter EL noted for highlevel expression.

A useful feature of vNotI/tk that must be retained in any new vector isthe ability to discriminate for recombinant viral genomes usingselection against an active thymidine kinase gene. The introduction ofthe ApaI site within the coding sequence for the tk gene necessitates anincrease in the total number of amino acids in order to accommodate therestriction enzyme site. A comparison of the amino acid sequence forthymidine kinase genes from a variety of animal and viral species showedthe region of greatest heterogeneity was at the N terminus of theprotein, suggesting that this region of the protein could tolerate amodest increase in the number of amino acids.

The recombination-independent cloning vectors were constructed by makingplasmid intermediates containing the modified thymidine kinase (tk) geneand replacing the tk sequence in the vNotI/tk genome by homologousrecombination. Two sets of oligonucleotide pairs were constructed which,when annealed, contained the promoter for the 7.5k gene or the syntheticEL sequence and restriction sites for NotI and ApaI. The modifiedthymidine kinase genes were constructed by annealing the double-strandedoligonucleotides and ligating the product into the NotI site present atthe beginning of the thymidine kinase gene in pJNotI/tk, a plasinidcontaining the HindIII J fragment from vNotI/tk. The oligonucleotidepairs annealed to and eliminated the NotI site in pJNotI/tk generating anew NotI site closely followed by an ApaI site after the promoter andflanking the nucleotides coding for the initial methionine in thethymidine kinase gene resulting in plasmids p7.5/tk (SEQ ID NO: 1) andpEL/tk (SEQ ID NO:3) (FIG. 1). The acquisition of the ApaI site wasverified by restriction enzyme analysis of plasmid DNA and thenucleotide sequence of the thymidine kinase gene promoter was determinedand found to be as depicted in FIG. 1.

The recombinant viruses derived from p7.5/tk and pEL/tk were isolatedusing a strategy relying on positive drug selection in the presence ofHAT (hypoxanthine, aminopterin, thymidine) (Weir, et al., Proc. Nat.Acad. Sci. USA 79:1210-1214 (1982)). The viruses vpNotI, a virus thatcontains a copy of pBR322 inserted at the NotI site of vNotI/tk(Merchlinsky, et al., Virology 190:522-526 (1992)), and vNotI/lacZ/tk, avirus with a copy of the lacZ gene interrupting the thymidine kinase invNotI (Merchlinsky, et al., Virology. 190:522-526 (1992)) are thymidinekinase negative (tk⁻) viruses that are identical to vNotI/tk except forthe inserted DNA at the beginning of the tk gene. The plasmids p7.5/tkand pEL/tk were recombined with vpNotI and vNotI/lacZ/tk helper virusesin CV-1 cells and the infected monolayers were harvested and passaged inthe presence of HAT media on Hutk⁻ cells. Individual plaques werepassaged and isolated an additional three rounds on Hutk⁻ cells beforeexpansion and analysis.

Analysis of the Structure of the Viral Genomes

The growth of v7.5/tk and vEL/tk virus in HAT supplemented media impliesthese viruses, in contrast to vpNot and vNot/lacZ/tk, contain an activethymidine kinase (tk) gene. However, an active tk gene could arise frommultiple crossovers which delete the 7.5k or EL promoter sequences,generating a virus with the normal tk promoter. The v7.5/tk and vEL/tkgenomes should contain a unique site for both NotI and ApaI within theHindIII J fragment. The genomic structure of the isolated virus stockswas analyzed by restriction enzyme digestion of DNA in agarose plugsderived from virus infected cells using NotI or ApaI and electrophoresisof the products through 1% agarose (FIG. 10). Uncut vaccinia WR (lane 2)migrates at a size of 190 kilobase pairs as compared to multimers ofbacteriophage lambda (lane 1). After digestion with NotI vaccinia WR iscleaved into two fragments approximately 150 and 40 kilobase pairs inlength (7th lane from left) whereas the vNot/tk, vEL/tk, and v7.5/tkwere cleaved into fragments of about 110 and 80 kilobase pairs. When thesame samples were digested with ApaI, only one fragment the size of theuncut genome was observed for both vaccinia WR and vNot/tk while vEL/tkand v7.5/tk gave the same sized fragments observed after digestion withNotI. Therefore, both v7.5/tk and vEL/tk contain a unique site for bothApaI and NotI, the sites are at the same locus as the NotI site invNot/tk, and the sites are in a more central location in the genome thanthe HindIII F fragment which contains the NotI site in vaccinia WR. Thebackground of cellular DNA fragments was more pronounced in the ApaIdigestion, which has a six base pair recognition site, than for the NotIdigest.

The genomes for vEL/tk and v7.5/tk were analyzed by Southern blotting toconfirm the location of the ApaI and NotI sites in the HindIII Jfragment as shown in FIGS. 11A and 11B. The filters were hybridized to³²P labeled HindIII J fragment derived from the p7.5/tk or pEL/tk. Thegenomes for v7.5/tk and vEL/tk have an ApaI site that does not appear invNotI/tk (compare lanes 7 and 8 to lane 5 in each blot) whereasdigestion with NotI and HindIII yield a set of fragments of equivalentsize. The 0.5 kilobase HindIII/NotI or HindIII/ApaI fragment from theleft hand side of HindIII J produced from NotI or ApaI digestion haselectrophoresed off the bottom of the agarose gel.

The definitive characterization of tile promoter sequence utilizedproducts of Polymerase Chain Reaction (PCR). A pair of primers flankingthe beginning of the tk gene were used to generate a DNA fragment fromthe viruses vNotI/tk, v7.5/tk, or vEL/tk and their cognate plasmids asshown in FIG. 12. The PCR products for v7.5/tk and vEL/tk are the samesize as those observed for the plasmids used to generate the viruses(p7.5/tk and pEL/tk) and larger than those seen for vaccinia WR andvNotI/tk. Tile PCR fragments were cloned into the plasmid pCRII, thenucleotide sequence was determined and shown to match the sequencedisplayed in FIG. 1.

Quantitation of Promoter Activity

The v7.5/tk and vEL/tk vectors have been designed to constitutivelyexpress elevated levels of insert protein in comparison to vNotI/tk. Thelevel of RNA synthesis was measured by infecting confluent BSC-1 cellsin the presence and absence of cytosine arabinoside (AraC) at an moi of5, harvesting the cells, isolating the RNA using Trizol (LifeTechnologies) and analyzing the level of thymidine kinase RNA synthesisby primer extension (Weir, et al., Nucleic Acids Research 16:10267-10282(1990)). Incubation with AraC blocks viral DNA replication, allowing oneto identify the class of viral promoter.

The early class of viral promoters are active prior to DNA replicationand will be unaffected by AraC in the infection. Late promoters are onlyexpressed after the onset of DNA replication and their activity isabrogated in the presence of AraC. Perusal of the products on adenaturing polyacrylamide gel demonstrated that significantly more(estimated to be at least ten fold) tk RNA primer extension productswere synthesized in vEL/tk infections as compared to vNot/tk. In cellsinfected with vNot/tk a single RNA start site insensitive to AraCincubation was observed whereas in vEL/tk infections two distinct startsites, one resistant to AraC and corresponding to the appropriate earlystart site (Davison, et al., J. Mol. Biol. 210:749-769 (1989)), and onespecies sensitive to AraC and corresponding to the appropriate latestart of RNA (Davison, et al. J. Mol. Biol. 210:771-784 (1989)) wereobserved (data not shown). The pattern of RNA species derived frominfection with v7.5/tk was similar to that observed for vEL/tk with theabsolute levels of RNA expression intermediate to that observed forvEL/tk and vNot/tk.

In order to verify the levels of expression for genes inserted into theviral vectors the E. coli gusA gene encoding for β-glucuronidase (β-glu)was cloned into vNotI/tk, v7.5/tk and vEL/tk viral vectors and therelative promoter strength was measured. The DNA fragment encoding forthe β-glu gene was inserted into plasmids containing each promotergenerating pJNot/tk-GUS, p7.5/tk-GUS and pEL/tk-GUS. The correctorientation of the insert β-glu gene in pJNot/tk was verified byrestriction enzyme analysis. The plasmids were recombined with vNotI/tkand the recombinant viruses identified by staining with X-glu (Carroll,et al., 1995, BioTechniques 19:352-355). passaged for three roundsthrough Hutk⁻ cells, and expanded to generate the viral stocksvNotI/tk-GUS, v7.5/tk-GUS and vEL/tk-GUS. The structures of therecombinant viruses were verified by Southern blot analysis.

The level of expression of β-glu by vNotI/tk-GUS, v7.5/tk-GUS andvEL/tk-GUS was measured from infected confluent monolayers of BSC-1cells in the presence or absence of AraC (FIG. 13). The level of β-gluexpression for the v7.5/tk-GUS and vEL/tk was much higher than thatobserved for vNotI/tk-GUS and highest (approximately twenty fold higher)in the vEL/tk-GUS. Expression of β-glu was observed for all threeviruses in the presence of cytosine arabinoside, indicating that eachpromoter is a member of the early class of viral promoters. The level ofβ-glu in vNotI/tk-GUS was unchanged in the presence or absence of AraCindicating that this promoter is only active early during infection,whereas the β-glu levels in v7.5/tk-GUS and vEL/tk-GUS were lower in thepresence of AraC, indicating these promoters are active both early andlate times during infection.

Biochemical Characterization of Virus Vectors

The v7.5/tk and vEL/tk vectors were initially isolated by growth in thepresence of HAT supplemented media and are designed to contain an activetk gene to allow selection for viruses with inserts via passage in Hutk⁻cells in the presence of bromodeoxyuridine (Earl, et al., in CurrentProtocols in Molecular Biology, Ausubel et al., eds., Greene PublishingAssociates/Wiley Interscience, New York (1991)). Both vectors weretested by plaque assay in Hutk⁻ cells using drug selection and theresults for vEL/tk are shown in FIG. 14. Incubation without drug or withHAT supplement at a concentration sufficient to interfere with plaqueformation for vpNot or vNot/lacZ/tk, (data not shown), gave anequivalent number of like-sized plaques. Surprisingly, an equal numberof plaques. albeit much smaller in size, were observed for vEL/tk withincubation in 25 mM bromodeoxyuridine, a concentration sufficient tointerfere with the ability of vaccinia WR to plaque on Hutk⁻ cells (datanot shown). Addition of 125 mM bromodeoxyuridine was sufficient toinhibit plaque formation for vEL/tk (FIG. 14) and v7.5/tk (data notshown). The higher concentration of bromodeoxyuridine did not interferewith the growth of tk⁻ viruses such as vNotI/lacZ/tk (data not shown) oraffect the viability of the Hutk⁻ cell line.

Construction of Recombinant Virus by Direct Ligation

Direct ligation vectors will only be useful for the generation ofcomplex expression libraries if the production of infectious virus fromthe naked DNA is facile and efficient. Previously, helper virus activitywas supplied in cells transfected with DNA ligation products bycoinfection with conditionally lethal temperature sensitive virus(Merchlinsky, et al., Virology. 190:522-526 (1992)) or fowlpox(Scheiflinger, et al., Proc. Natl. Acad. Sci. USA 89:9977-9981 (1992)).Since high levels of replicating wild type virus interfere with theability to package viral DNA and vaccinia virus can recombine with theinput DNA, only conditionally defective vaccinia virus can be used ashelper (Merchlinsky, et al., Virology 190:522-526 (1992)). Fowlpoxshould be a superior helper virus as it is used at 37° C., will notrevert to a highly replicating strain, and, since it does not recombinewith vaccinia DNA or productively infect primate cell lines, can be usedat higher moi than vaccinia. In order to determine if fowlpox can serveas an efficient helper virus a series of wells from a 12 well platecontaining BSC-1 cells were infected with varying mois of fowlpox andtransfected with full length vaccinia WR DNA, the cells were harvestedafter 24, 48, or 72 hours and the virus titer was determined as shown inTable 8. Transfection of DNA sans fowlpox or fowlpox infection aloneresulted in no plaques. The level of rescued vaccinia increased withlater harvest and was proportional to the moi of the fowlpox infection.

TABLE 8 FPV moi Day harvested Titer (pfu × 10⁻³) 0.2 1 0 2 0.12 3 3000.5 1 0 2 0.23 3 500 1.0 1 0 2 1.1 3 700

Table 8. Packaging of vaccinia DNA by fowlpox virus. Vaccinia DNA wastransfected into BSC-1 cells infected with fowlpox virus usinglipofectamine as described in Example 9 (Materials and Methods). Thecells were harvested at 1, 2, or 3 days post transfection, lysed byfreeze-thaw cycles and assayed for infectious virus by plaque assay onBSC-1 cells.

A 1.1 kilobase pair fragment of the ovalbumin cDNA (Pulaski, et al.,Proc. Natl. Acad Sci. USA 93:3669-3674 (1996)) was used as a modelinsert to study the generation of functional recombinant virus by directligation. The ovalbumin insert was modified as described in theMaterials and Methods to include a NotI site at its 5′ end, translationstop codons, a vaccinia transcription stop signal and an ApaI site atits 3′ end. This insert was digested with NotI and ApaI and ligated withpurified vEL/tk DNA arms that had been digested with NotI and ApaI. Theligation mix was transfected into fowlpox infected BSC-1 cells, cellswere harvested, and after three days the cell extract was passaged onHutk⁻ cells in the presence or absence of 125 mM bromodeoxyuridine. Thetiter obtained without drug selection was 2.7×10³ pfu and with drugselection 2.8×10³ pfu. Individual plaques were picked from Hutk⁻cells inthe presence and absence of bromodeoxyuridine and tested for thepresence of the ovalbumin insert by dot blot hybridization with anovalbumin cDNA probe. All 15 plaques picked in the presence ofbromodeoxyuridine, and all 10 plaques picked in its absence containedthe ovalbumin insert. These viruses were named vEL/tk-ovaFL. Twoindividual clones were expanded further and tested for the ability tosensitize host cells to lysis by ova 257-264 specific cytotoxic Tlymphocytes (CTL). The results of this experiment are shown in Table 9.As controls, vaccinia recombinant for an ova 257-264 minigene,v7.5/tk-ova and vEL/tk-ova, were generated by homologous recombination.These ova peptide recombinant viruses were tested in concert with thevEL/tk-ovaFL clones for the ability to sensitize host cells to lysis byova specific CTL. As shown in Table 9, infection with either full lengthor minigene ovalbumin vaccinia recombinants was as efficient as pulsingwith 1 μM purified OVA 257-264 peptide for sensitization of target cellsto lysis by OVA-specific CTL.

TABLE 9 Effector:Target Ratio 2:1 10:1 MC57G cells: (Percent SpecificLysis) Untreated −1.3 −1.3 ova257-264 peptide, 1 μM 54 83 vEL/tk −0.5 0v7.5/tk-ova Homologous 50 78 Recombination vEL/tk-ova Homologous 47 71Recombination vEL/tk-ovaFL Direct Ligation Clone 1 48 70 vEL/tk-ovaFLDirect Ligation Clone 2 46 74

Table 9. CML assay on recombinant vaccinia virus infected cells. Virallyinfected MC57G cells were generated as described in Example 9 (Materialsand Methods). One sample of MC57G cells was treated with ova257-264peptide (1 μM), another sample of cells was left untreated. Cells wereincubated with two different ratios of ova specific cytotoxic Tlymphocytes for 4 hours at 37° C. and percent specific lysis wasdetermined as described in Example 9 (Materials and Methods).

Discussion

Large DNA viruses are particularly useful expression vectors for thestudy of cellular processes as they can express many different proteinsin their native form in a variety of cell lines. In addition, geneproducts expressed in recombinant vaccinia virus have been shown to beefficiently processed and presented in association with MHC class I forstimulation of cytotoxic T cells. The gene of interest is normallycloned in a plasmid under the control of a promoter flanked by sequenceshomologous to a non-essential region in the virus and the cassette isintroduced into the genome via homologous recombination. A panoply ofvectors for expression, selection and detection have been devised toaccommodate a variety of cloning and expression strategies. However,homologous recombination is an ineffective means of making a recombinantvirus in situations requiring the generation of complex libraries orwhen the insert DNA is large. An alternative strategy for theconstruction of recombinant genomes relying on direct ligation of viralDNA “arms” to an insert and the subsequent rescue of infectious virushas been explored for the genomes of poxvirus (Merchlinsky, et al.,Virology 190:522-526 (1992); Pfleiderer, et al., J. General Virology76:2957-2962 (1995); Scheiflinger, et al., Proc. Natl. Acad. Sci. USA89:9977-9981 (1992)), herpes virus (Rixon, et al., J. General Virology71:2931 -2939 (1990)) and baculovirus (Ernst, et al., Nucleic AcidsResearch 22:2855-2856 (1994)).

Poxviruses are ubiquitous vectors for studies in eukaryotic cells asthey are easily constructed and engineered to express foreign proteinsat high levels. The wide host range of the virus allows one tofaithfully express proteins in a variety of cell types. Direct cloningstrategies have been devised to extend the scope of applications forpoxvirus viral chimeras in which the recombinant genomes are constructedin vitro by direct ligation of DNA fragments to vaccinia “arms” andtransfection of the DNA mixture into cells infected with a helper virus(Merchlinsky, et al., Virology 190:522-526(1992); Scheiflinger, et al.,Proc. Natl. Acad. Sci. USA 89:9977-9981 (1992)). This approach has beenused for high level expression of foreign proteins (Pfleiderer, et al.,J. Gen. Virology 76:2957-2962 (1995)) and to efficiently clone fragmentsas large as 26 kilobases in length (Merchlinsky, et al., Virology190:522-526 (1992)).

Vaccinia virus DNA is not infectious as the virus cannot utilizecellular transcriptional machinery and relies on its own proteins forthe synthesis of viral RNA. Previously, temperature sensitiveconditional lethal (Merchlinsky, et al., Virology 190:522-526 (1992)) ornon-homologous poxvirus fowlpox (Scheiflinger, et al., Proc. Natl. Acad.Sci. USA 89:9977-9981 (1992)) have been utilized as helper virus forpackaging. An ideal helper virus will efficiently generate infectiousvirus but not replicate in the host cell or recombine with the vacciniaDNA products. Fowlpox virus has the properties of an ideal helper virusas it is used at 37° C., will not revert to a highly replicating strain,and, since it does not recombine with vaccinia DNA or productivelyinfect primate cell lines, can be used at relatively high moi.

The utility of the vaccinia based direct ligation vector vNotI/tk, hasbeen described by Merchlinsky, et al., Virology 190:522-526 (1992)).This genome lacks the NotI site normally present in the HindIII Ffragment and contains a unique NotI site at the beginning of thethymidine kinase gene in frame with the coding sequence. This allows theinsertion of DNA fragments into the NotI site and the identification ofrecombinant genomes by drug selection. The vNotI/tk vector can be usedto efficiently clone large DNA fragments but does not fix theorientation of the DNA insert or lead to high expression of the foreignprotein. This example describes the construction and characterization ofa pair of vaccinia DNA vector genomes v7.5/tk and vEL/tk suitable fordirect ligation. The v7.5/tk and vEL/tk vectors were designed to containunique restriction sites for NotI and ApaI at the beginning of thethymidine kinase gene allowing the oriented cloning of DNA andeliminating the intact genomes arising from relegation of vacciniavector arms.

The vNotI/tk vector will only express foreign proteins at the level ofthe thymidine kinase gene, a weakly expressed gene only made earlyduring viral infection. To induce high levels of protein expression thesequences encoding for the viral 7.5k promoter and a synthetic ELpromoter devised by Chakrabarti and Moss were used to replace theendogenous thymidine kinase promoter. The levels of expression inducedby either promoter was much higher than that observed in vNotI/tk andthe promoters were active at all times post infection. These continuousexpression vectors are applicable in cases dependent on earlyexpression, such as T-cell epitope presentation, as well as for bulkexpression of proteins.

Use of the thymidine kinase gene as the insertion site for foreign DNAallows implementation of selection protocols for distinguishingrecombinants from helper or wild type genomes. The level of tkexpression in v7.5/tk and vEL/tk should be much higher than in vacciniaWR or vNot/tk. However, the ApaI site at the beginning of the tk gene inv7.5/tk and vEL/tk was formed from vNot/tk by adding extra nucleotidesat the NotI site. The additional nucleotides increase the amino acidsequence at the N terminus of the wild type tk gene from Met-Asn-Gly toMet-Gly-Pro-Ala-Ala-Asn-Gly in v7.5/tk and vEL/tk. Modifications in theexpression level and N terminal amino acid sequence of the thymidinekinase gene may increase (more protein) or decrease (different sequence)the sensitivity of the virus to bromodeoxyuridine. Plaques, albeitsmaller, were observed with v7.5/tk and vEL/tk infection at aconcentration of bromodeoxyuridine sufficient to completely suppressplaque formation for wild type vaccinia WR. Plaque formation wassuppressed at five-fold higher concentrations of bromodeoxyuridine, alevel of drug that does not interfere with the viability of the cells orimpede the ability of tk⁻ virus to form plaques. The explanation for thealtered sensitivity to bromodeoxyuridine awaits further characterizationof the protein as the altered thymidine kinase gene may have a differentreaction rate for formation of the triphosphate form of thebromodeoxyuridine or a reduced ability to bind bromodeoxyuridine.

The development of direct ligation vectors has increased the possibleapplications for poxvirus expression vectors. The v7.5/tk and vEL/tkvectors were designed to incorporate the advantages of oriented cloning,high levels of expression of foreign protein, and the selection forrecombinant viruses, into direct ligation vectors. They were shown toexpress high levels of proteins at all times during infection. Theutility of these vectors was demonstrated by constructing recombinantscontaining a CTL epitope for ovalbumin (constructed by homologousrecombination with a plasmid) or the ovalbumin coding sequence(constructed by direct ligation protocol) and showing how bothrecombinants were able to elicit a strong CTL response

The application of these vectors to protocols for construction ofcomplex expression libraries requires efficient production ofrecombinants and strong selection to eliminate or minimize wild type andcontaminants. The use of two restriction sites allows one to designcloning strategies for the oriented cloning of DNA fragments such asproducts of PCR (Pfleiderer, et al., J. General Virology 76:2957-2962(1995)) and increases the frequency of the desired recombinant as wildtype genomes can no longer be generated by ligation of vaccinia arms.When v7.5/tk or vEL/tk DNA previously digested with NotI and ApaI wastransfected into cells infected with fowlpox the virus titer was onehundred fold lower than for intact uncut DNA. Also, all plaques isolatedin the presence and absence of bromodeoxyuridine (15 withbromodeoxyuridine and 10 without) during the isolation of thevEL/tk-ovaFL contained the ovalbumin insert. The efficiency ofinfectious virus formation is also increased with the use of fowlpox,helper virus at relatively high moi. Also, transfection of large DNAfragments varies with the type and preparation of lipid (Miles Carrollpersonal communication) and we are presently assaying different lipidmixtures and cell types as well as investigating other parameters tofind optimum conditions for the direct ligation protocol. The v7.5/tkand vEL/tk vectors provide a set of universally applicable directligation cloning vectors for poxviruses.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any constructs, viruses orenzymes which are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method for selecting a nucleic acid molecule encoding a targetepitope of cytotoxic T-lymphocytes, comprising: (a) contacting mammalianhost cells with cytotoxic T-lymphocytes specific for said target epitopeunder conditions wherein a host cell expressing said target epitopeundergoes a lytic event upon contact with said T-lymphocytes; whereinsaid host cells comprise a library of heterologous nucleic acidmolecules, at least one of said heterologous nucleic acid moleculesencoding said target epitope, wherein said library is constructed in avaccinia virus vector which expresses said target epitope in said hostcells, wherein said host cells express a defined MHC molecule, andwherein said cytotoxic T-lymphocytes are restricted for said MHCmolecule; (b) recovering the floating host cells which are undergoing alytic event, and (c) isolating said vector from said recovered hostcells, thereby selecting a nucleic acid molecule which encodes saidtarget epitope.
 2. The method of claim 1, further comprising: (d)transferring said vector to a population of mammalian host cells,wherein said vector expresses said target epitope in said host cells,and wherein said host cells express a defined MHC molecule; (e)contacting said host cells with cytotoxic T-lymphocytes specific forsaid target epitope and restricted for said MHC molecule, underconditions wherein a host cell expressing said target epitope willundergo a lytic even upon contact with said T-lymphocytes; (f)recovering the floating host cells which are undergoing a lytic event,and (g) isolating said vector from said recovered host cells, therebyselecting a nucleic acid molecule which encodes said target epitope. 3.The method of claim 1, wherein said vector further comprises atranscriptional control signal in operable association with saidheterologous nucleic acid molecules, and wherein said transcriptionalcontrol signal functions in a vaccinia virus.
 4. The method of claim 3,wherein said transcriptional control signal comprises a promoter.
 5. Themethod of claim 4, wherein said promoter is constitutive.
 6. The methodof claim 4, wherein said promoter is a vaccinia virus p7,5 promoter. 7.The method of claim 6, wherein said vector comprises the sequence shownin SEQ ID NO:1.
 8. The method of claim 4, wherein said promoter is asynthetic early/late promoter.
 9. The method of claim 8, wherein saidvector comprises the sequence shown in SEQ ID NO:3.
 10. The method ofclaim 3, wherein said transcriptional control signal comprises atranscriptional termination signal.
 11. The method of claim 3, whereinsaid vector further comprises a translational control signal associatedwith said transcriptional control signal.
 12. The method of claim 11,wherein said vector comprises the sequence shown in SEQ ID NO:6.
 13. Themethod of claim 11, wherein said translational control signal comprisesa translation initiation codon operably linked to said heterologousnucleic acid molecules.
 14. The method of claim 13, wherein saidtranslation initiation codon occurs in one of three reading frames. 15.The method of claim 14, wherein said vector comprises a sequenceselected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQID NO:9.
 16. The method of claim 1, wherein said library of heterologousnucleic acid molecules is isolated from a tumor cell, and wherein saidtarget epitope is differentially expressed in said tumor cell relativeto a non-tumorigenic counterpart cell.
 17. The method of claim 16,wherein said heterologous nucleic acid molecules are cDNA moleculessynthesized from said tumor cell.
 18. The method of claim 1, whereinsaid library is constructed by a method comprising: (a) cleaving avaccinia virus genome to produce a first viral fragment and a secondviral fragment, wherein said first fragment is nonhomologous with saidsecond fragment; (b) providing a population of transfer plasmidscomprising said heterologous nucleic acid molecules flanked by a 5′flanking region and a 3′ flanking region, wherein said 5′ flag region ishomologous to said first viral fragment and said 3′ flanking region ishomologous to said second viral fragment; and wherein said transferplasmids are capable of homologous recombination with said first andsecond viral fragments such that a viable virus genome is formed; (c)introducing said transfer plasmids and said first and second vialfragments into a host cell under conditions wherein a transfer plasmidand said viral fragments undergo in vivo homologous recombination,thereby producing a viable modified virus genome comprising aheterologous nucleic acid molecule; and (d) recovering said modifiedvirus genome.
 19. The method of claim 18, wherein said virus genomecomprises a first recognition site for a first restriction endonucleaseand a second recognition site for a second restriction endonuclease; andwherein said first and second viral fragments are produced by digestingsaid viral genome with said first restriction endonuclease and saidsecond restriction endonuclease, and isolating said first and secondviral fragments.
 20. The method of claim 19, wherein said first andsecond recognition sites are physically arranged in said genome suchthat the region extending between said first and second viral fragmentsis not essential for virus infectivity.
 21. The method of claim 18,wherein said transfer plasmids and said first and second viral fragmentsare introduced into a host cell comprising a helper virus, wherein saidhost cell is non-permissive for the production of infectious virusparticles of said helper virus.
 22. The method of claim 21, wherein saidhelper virus is an avipoxvirus.
 23. The method of claim 22, wherein saidavipoxvirus is a fowlpox virus.
 24. The method of claim 19, wherein saidfirst and second restriction enzyme recognition sites are situated in athymidine kinase gene.
 25. The method of claim 19, wherein said firstand second restriction enzyme recognition sites are situated in avaccinia virus HindIII J fragment.
 26. The method of claim 25, whereinsaid first restriction enzyme is NotI, and wherein said firstrestriction enzyme recognition site is GCGGCCGC.
 27. The method of claim25, wherein said second restriction enzyme site is ApaI, and whereinsaid second restriction enzyme recognition site is GGGCCC.
 28. Themethod of claim 18, wherein said vaccinia virus genome comprises amodified thymidine kinase (tk) gene which comprises a 7.5k promoter, aunique NotI restriction site, and a unique ApaI restriction site. 29.The method of claim 18, wherein said vaccinia virus genome comprises amodified thymidine kinase (tk) gene which comprises a syntheticcarly/late (E/L) promoter, a unique NotI restriction site, and a uniqueApaI restriction site.
 30. The method of claim 18, wherein the 5′ and 3′flanking regions of said transfer plasmids are capable of homologousrecombination with a vaccinia virus thymidine kinase gene.
 31. Themethod of claim 30, wherein the 5′ and 3′ flanking regions of saidtransfer plasmids are capable of homologous recombination with avaccinia virus HindIII J fragment.
 32. The method of claim 30, whereinsaid transfer plasmids comprise heterologous nucleic acid moleculesligated into a plasmid selected from the group consisting of: (a)p7.5/ATG0/tk, which comprises SEQ ID NO:6; (b) p7.5/ATG1/tk, whichcomprises SEQ ID NO:7; (c) p/7,5/ATG2/tk, which comprises SEQ ID NO:8;and (d) p7.5/ATG3/tk, which comprises SEQ ID NO:9.
 33. The method ofclaim 1, wherein said host cells are a monolayer, and wherein thefloating host cells which are undergoing a lytic event are released fromsaid monolayer.
 34. The method of claim 1, wherein said MHC molecule isa class I MHC molecule.
 35. The method of claim 2, wherein said hostcells are a monolayer, and wherein the floating host cells which areundergoing a lytic event are released from said monolayer.
 36. Themethod of claim 2, wherein said MHC molecule is a class I MHC molecule.